regulation of the dopamine d1-d2 receptor heterooligomer
TRANSCRIPT
Regulation of the Dopamine D1-D2 Receptor Heterooligomer
by
Vaneeta Verma
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Pharmacology and Toxicology University of Toronto
© Copyright by Vaneeta Verma (2011)
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Regulation of the Dopamine D1-D2 Receptor Heterooligomer
Vaneeta Verma
Doctor of Philosophy
Department of Pharmacology and Toxicology
University of Toronto
2011
ABSTRACT
Dopamine receptors are members of the G protein-coupled receptor superfamily and play
important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G-
protein linked PLC-dependent intracellular calcium signal was previously identified. The
discovery of this dopamine mediated calcium signal implicated a direct link between dopamine
receptors and calcium generation, but its regulation remained to be elucidated. By measuring
calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the
calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with
SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only
activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2
receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that
occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes
sufficient for desensitization without activation of the heteromer. BRET and co-
immunoprecipitation studies indicated an agonist induced interaction between the D1-D2
receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium
signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the
catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the
GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2
receptor heteromer mediated calcium signal can occur by agonist occupancy even without
activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and
calcium assays demonstrated that recycling of internalized D1 and D2 receptors and
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resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not
SKF 83959, suggesting that the trafficking and resensitization response associated with the D1-
D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results
suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent
receptors which are not coupled to Gq.
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ACKNOWLEDGEMENTS First and foremost, I would like to extend my sincerest gratitude to my supervisor, Dr.
Susan George. The outstanding mentorship I received from her over the years has taught me
valuable lessons that I will carry forward throughout my career. Her expertise and guidance have
been instrumental to my successes that I have achieved during my doctoral studies and have
helped me attain skills that are key for becoming a strong, independent scientist. Dr. George has
been an inspiration to me and working under her supervision has been an honor and a privilege. I
would also like to thank my co-supervisor, Dr. Brian O’Dowd. I appreciate his profound insight,
mentorship and guidance that have been immensely beneficial to this work.
Thank you to the funding agencies that helped support this research. These include the
Natural Sciences and Engineering Research Council of Canada, Peterborough K.M Hunter
Graduate Scholarship, Canadian Institutes of Health Research and the National Institutes of
Health.
I would also like to thank my committee members, Dr. Denis Grant and Dr. Peter
McPherson, as well as my examination committee, Dr. Ali Salahpour, Dr. Denis Grant, Dr. Scott
Heximer, Dr. Jose Nobrega, and Dr. Stephen Ferguson for their valuable advice, insightful
comments and providing different perspectives on this work.
A special thanks to all of the members of the lab for creating a friendly and motivating
environment to work in. Specifically, Theresa Fan, Tuan Nguyen, and Tony ji for providing me
with exceptional technical assistance. Their skills, time and energy are highly appreciated and
greatly contributed to this work. I would also like to send a special thanks to Dr. Melissa
Perreault, Dr. Ahmed Hasbi, Dr. Christopher So, Dr. Asim Rashid, and Dr. Michael Kong. They
have been my mentors and friends, always giving me sound advice and never hesitating to
provide either an ear to listen or a hand to help. I am very grateful for their efforts which were
pivotal to the success of this work.
I would like to thank my family for their constant patience and encouragement and
always believing in me.
Last but not least, I would like to thank my friends for always being there for me in good
times and in bad, helping make my time at graduate school fun and enjoyable and providing
great memories that will last a life time.
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TABLE OF CONTENTS ABSTRACT ii
AKNOWLEDGEMENTS iv
TABLE OF CONTENTS v
LIST OF PUBLICATIONS viii
LIST OF FIGURES ix
SUMMARY OF ABBREVIATIONS xii
1 INTRODUCTION 1
1.1 Overview of G protein-Coupled Receptors 1 1.1.1 G Proteins 2 1.2 Control of Receptor Signaling 4 1.2.1 Agonist-dependent GPCR Desensitization 4 1.2.2 Agonist-dependent GPCR Internalization 7 1.2.3 GPCR Recycling and Degradation 9 1.3 Introduction to GPCR Oligomerization 10 1.3.1 Evidence for GPCR Oligomerization 11 1.3.2 GPCR Stoichiometry 12 1.3.3 Structural Features of GPCR Oligomers 14 1.4 Regulation of GPCR Oligomers 16 1.4.1 Agonist-dependent Desensitization of GPCR Oligomers 16 1.4.2 Agonist-dependent Internalization of GPCR Oligomers 17 1.4.3 Post Endocytic Sorting of GPCR Oligomers 19 1.4.4 Stability of GPCR Oligomers 20 1.5 Dopamine 21 1.5.1 Dopamine Pathways and Functions 22 1.6 Dopamine Receptors 23 1.7 Dopamine D1 Receptors 24 1.7.1 Cellular Signaling of D1 Receptors 24 1.7.2 Desensitization of D1 Receptors 27 1.7.3 Internalization of D1 Receptors 29 1.7.4 Resensitization and Recycling of D1 Receptors 32 1.7.5 D1 Receptor Homooligomers and Heterooligomers 33 1.8 Dopamine D2 Receptors 35 1.8.1 Cellular Signaling of D2 Receptors 35 1.8.2 Desensitization of D2 Receptors 35 1.8.3 Internalization of D2 Receptors 37
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1.8.4 Resensitization and Recycling of D2 Receptors 39 1.8.5 D2 Receptor Homooligomers and Heterooligomers 39 1.9 The D1-D2 Receptor Heterooligomer 41 1.9.1 D1-D2 Receptor Heterooligomers in vitro and in vivo 42 1.9.2 Activation of the D1-D2 Receptor Heterooligomer in Striatum 43 1.9.3 Functional Consequences of D1-D2 Receptor Heterooligomer 44 Mediated Signaling 1.9.4 Pharmacology of the D1-D2 Receptor Heterooligomer 46 1.9.5 D1-D2 Receptor Heterooligomer Desensitization 49 1.9.6 D1-D2 Receptor Heterooligomer Internalization 50 1.10 Research Rationale and Objectives 50
2 MATERIALS AND METHODS 53
2.1 Cell Culture 53 2.2 Transient Transfections in HEK293T Cells 53 2.3 Measurement of the Calcium Signal in HEK293T Cells 54 2.4 Membrane Preparation and Radioligand Saturation Binding Assay 55 2.5 Intact Cell Radioligand Binding Assay 56 2.6 Immunocytochemistry of HEK293T Cells 56 2.7 Neuronal Cultures 57 2.8 Immunocytochemistry of Cultured Neurons 58 2.9 Confocal Microscopy FRET 58 2.10 Measurement of the Calcium Signal in Primary Striatal Neurons 59 2.11 Immunoprecipitation 61 2.12 BRET Assay 62 2.13 SDS-Polyacrylamide Gel Electrophoresis 62 2.14 Statistical Analysis 63 3 RESULTS 64 3.1 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal 64 in HEK293T Cells 3.2 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 64 in HEK293T Cells 3.2.1 Desensitization Through Selective Occupancy 71 of the D1 Receptor 3.3 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal 74 in Primary Striatal Neurons 3.4 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 77 in Primary Striatal Neurons 3.5 Role of GRK2 in Regulating the D1-D2 Receptor Heteromer Mediated 80 Calcium Signal 3.5.1 Evaluation of GRK2 Functional Domains in Regulating 80 the D1-D2 Receptor Heteromer Mediated Calcium Signal
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3.5.2 Knockdown of GRK2 in HEK293T Cells and Striatal Neurons 85 3.5.3 D1-D2 Receptor Heteromer Interaction with GRK2 85 3.6 Resensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 92 in HEK293T Cells 3.7 Internalization and Recycling of D1 and D2 Receptors Following Treatment 92 with Dopamine or SKF 83959
4 DISCUSSION 98
4.1 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 98 4.2 Internalization of the D1-D2 Receptor Heteromer and Resensitization of the 105 Associated Calcium Signal 4.3 Related Studies 109 4.4 Novel Findings and General Conclusions 110 4.5 Significance and Future Studies 115 4.6 Concluding Remarks 124 5 REFERENCES 125
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LIST OF PUBLICATIONS 1. Kong MM*, Verma V*, O’Dowd BF, George SR (2011). The role of palmitoylation in directing
dopamine D1 receptor internalization through selective endocytic routes. Biochem Biophys Res Commun. 405:445-9 * These authors contributed equally to this work
2. Verma V, Hasbi A, O’Dowd BF, George SR (2010). Dopamine D1-D2 receptor heteromer-mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2. J Biol Chem. 285:35092-103.
3. Perreault M*, Verma V*, O’Dowd BF, George SR (2009). Regulation of dopamine receptor trafficking and responsiveness. (Review) The Dopamine Receptors, Second Edition.
* These authors contributed equally to this work
4. So CH, Verma V, Alijaniaram M, Cheng R, Rashid AJ, O'Dowd BF, George SR (2009). Calcium signaling by dopamine D5 receptor and D5-D2 receptor heterooligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor heterooligomers. Mol Pharmacol 75: 843-54.
5. So CH, Verma V, O’Dowd BF, George SR (2007). Desensitization of the dopamine D1 and D2 receptor hetero-oligomer mediated calcium signal by agonist occupancy of either receptor. Mol Pharmacol 72:450-62.
6. Rashid AJ, O’Dowd BF, Verma V, George SR (2007). Neuronal Gq/11 coupled dopamine receptors: an uncharted role for dopamine (Review). Trends in Pharmacology 28:551-5.
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LIST OF FIGURES Figure 1-1. Schematic diagram of the classical regulation of GPCR signaling. 6
Figure 1-2. General structure of a GRK protein. 8
Figure 1-3. Schematic diagram of the dopamine D1 receptor. 25
Figure 1-4. Schematic diagram of the dopamine D2 receptor. 26
Figure 1-5. Differential agonist activation of the D1-D2 receptor 48
heterooligomer and D1 receptor homooligomer Figure 3-1. Specificity of dopamine receptor agonists activating the 65
D1-D2 receptor heteromer calcium signal in cells stably expressing the D1 and D2 receptors.
Figure 3-2. The D1-D2 receptor heteromer mediated calcium signal is 67
desensitized by prior treatment with dopamine agonists for 5 min. Figure 3-3. The D1-D2 receptor heteromer mediated calcium signal is 68
desensitized by prior treatment with dopamine agonists for 10 min. Figure 3-4. The D1-D2 receptor heteromer mediated calcium signal is 69
desensitized by prior treatment with dopamine agonists for 30 min. Figure 3-5. The ATP induced calcium signal is not reduced by prior treatment 70
with agonist, SKF 83959 Figure 3-6. Effect of the adenylyl cyclase inhibitor, SQ 22536, on the SKF 83822 72
induced desensitization of the D1-D2 receptor heteromer mediated calcium signal.
Figure 3-7. Effect of dopamine receptor antagonists on the desensitization 73
of the D1-D2 receptor heteromer mediated calcium signal in D1-D2 receptor stably expressing cells.
Figure 3-8. Effect of D2 receptor agonist, quinpirole, on the desensitization 75
of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors.
Figure 3-9. Specificity of dopamine receptor agonists activating the 76
D1-D2 receptor heteromer calcium signal in primary striatal neurons.
x
Figure 3-10. The D1-D2 receptor heteromer mediated calcium signal is desensitized 78 in striatal neurons by prior treatment with dopamine agonists for 30 min.
Figure 3-11. Effect of dopamine receptor antagonists on the desensitization 79
of the D1-D2 receptor heteromer mediated calcium signal in striatal neurons.
Figure 3-12. Increased expression of GRK2 led to a concentration dependent 81
decrease of the D1-D2 receptor heteromer activated calcium signal. Figure 3-13. Effect of catalytic domain mutated or RGS domain mutated GRK2 83
on the D1-D2 receptor heteromer mediated calcium signal. Figure 3-14. Effect of GRK2, catalytic domain mutated or RGS domain mutated 84
GRK2 on the D1-D2 receptor heteromer mediated calcium signal after agonist pretreatment with dopamine agonists for 30 min.
Figure 3-15. Decreased expression of GRK2 by siRNA led to significant 86
recovery of the D1-D2 receptor heteromer mediated calcium signal after pretreatment with either SKF 83959 or SKF 83822 in the HEK 293T D1-D2 receptor heteromer stable cell line.
Figure 3-16. Decreased expression of GRK2 by siRNA led to significant 87
recovery of the D1-D2 receptor heteromer mediated calcium signal after pretreatment with either SKF 83959 or SKF 83822 in striatal neurons.
Figure 3-17. Immunocytochemistry of striatal neurons in culture 88
showing endogenously expressed GRK2 localization before and after exposure to either 100nM dopamine, SKF 83959 or SKF 83822 for 5 min.
Figure 3-18. Co-immunoprecipitation of HA-D1 receptor and GRK2 90
with Flag-D2 receptor from P2 membranes expressing Flag-D2 receptor, HA-D1 receptor and GRK2 after the HEK 239T cells were treated with vehicle, 1 μM dopamine, SKF 83959, or SKF 83822 for 5 min.
Figure 3-19. BRET detection of Rluc-D1 and GFP-GRK2 interaction after 91
either 1 or 10 min treatment with either vehicle, 1 μM dopamine, SKF 83959, or SKF 83822.
Figure 3-20. Resensitization of the D1-D2 receptor heteromer mediated 93
calcium signal in HEK 293T cells stably expressing D1 and D2 receptors.
Figure 3-21. Trafficking of the D1 and D2 receptors after treatment with 94
dopamine in HEK 293T cells stably expressing D1 and D2 receptors.
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Figure 3-22. Trafficking of the D1 and D2 receptors after treatment with 95
SKF 83959 in HEK 293T cells stably expressing D1 and D2 receptors..
Figure 3-23. Agonist induced internalization of the D1 receptor in HEK 293T 97 cells expressing the D1 receptor alone or co-expressing both the D1 and D2 receptors.
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SUMMARY OF ABBREVIATIONS AFM Atomic Force Microscopy
AFU Absolute Fluorescence Units
AMPH Amphetamine
ATP Adenosine Triphosphate
BDNF Brain Derived Neurotrophic Factor
BRET Bioluminescence Resonance Energy Transfer
CaMKII Ca2+ / Calmodulin Dependent Kinase II
cAMP Cyclic Adenosine Monophosphate
cDNA Complementary Deoxyribonucleic Acid
CFP Cyan Fluorescent Protein
CHO Chinese Hamster Ovary
D2L Dopamine D2 Long
D2S Dopamine D2 Short
ECL Enhanced Chemiluminescence
EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid
FRET Fluorescence Resonance Energy Transfer
FRAP Fluorescence Recovery After Photobleaching
GABA γ-aminobutyric acid
GASP G Protein Coupled Receptor Associated Sorting Protein
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GDP Guanosine Diphosphate
GFP Green Fluorescent Protein
GPCR G Protein-Coupled Receptor
GRK G Protein Receptor Kinase
GTP Guanosine Triphosphate
HA Hemagglutinin
HBSS HEPES-buffered Saline
HEK Human Embryonic Kidney
HEPES 4-(2-hydroxyethyl)-1-Piperazineethanesulfonic Acid
H89 N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline
IC3 Intracellular Loop
IP3 Inositol 1,4,5 Trisphosphate
L-Dopa L-dihydroxyphenlalanine
MAPK Mitogen-activated Protein Kinase
PBS Phosphate Buffered Saline
PH Pleckstrin Homology
PKA Protein Kinase A
PKC Protein Kinase C
PMCAs Plasma Membrane Calcium-ATPases
PLC Phospholipase C
PSD-95 Post Synaptic Density-95
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PTX Pertussis Toxin
RGS Regulator of G Protein Signaling
Rluc Renilla luciferase
SDS Sodium Dodecyl Sulfate
SKF 81297 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol
SKF 83822 ([R/S]-6-chloro-7, 8-dihydroxy-3-allyl-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-
1H-3-benzazepine)
SKF 83959 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-
benzazepine
SEM Standard Error of the Mean
siRNA Small Interfering Ribonucleic Acid
SQ 22536 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine
TM Transmembrane Domain
YFP Yellow Fluorescent Protein
VTA Ventral Tegmental Area
ZIP Zeta interacting protein
1
1 INTRODUCTION
1.1 Overview of G protein-Coupled Receptors
Heptahelical G protein-coupled receptors (GPCRs) form a large superfamily of cell-
surface receptors that respond to a diverse array of sensory and chemical stimuli, such as light,
odors, hormones and neurotransmitters. There are over 800 GPCRs in the human genome
making them the largest superfamily of plasma membrane proteins involved in signal
transduction (Lagerstrom and Schioth, 2008).
All GPCRs have a structurally similar core of seven transmembrane (TM) α helices,
three intracellular loops, three extracellular loops, an amino terminal extracellular domain and a
carboxyl terminal intracellular domain. Crystallography of the prototypical family 1 GPCR,
rhodopsin, in 2000 represented a milestone in the GPCR field and revealed that the TMs are
arranged in a ring-like fashion, forming a tightly packed helical bundle (Palczewski et al., 2000).
In the last ten years several other GPCRs have been crystallized, including the human β2
adrenergic (Cherezov et al., 2007; Rasmussen et al., 2007), turkey β1 adrenergic (Warne et al.,
2008), human A2A adenosine (Jaakola et al., 2008), human CXCR4 chemokine (Wu et al., 2010)
and the human D3 dopamine receptor (Chien et al., 2010).
The GPCR superfamily is classified into six main families or classes determined by
protein sequence similarity (Davies et al., 2007). These include family 1 or class A rhodopsin
like, family 2 or class B secretin like, family 3 or class C metabotropic glutamate, family 4 or
class D pheromone, family 5 or class E cAMP, and family 6 or class F frizzled/smoothened
receptors. Family 1 is the largest and most studied of the classes, accounting for ~80% of the
entire GPCR superfamily. Family 2 is much smaller with approximately 53 receptors, including
2
receptors for the gastrointestinal peptide hormone family, corticotrophin-releasing hormone,
calcitonin and parathyroid hormone. Family 3 has only approximately 19 receptors including γ-
aminobutyric acid (GABA) receptors, metabotropic glutamate receptors, calcium sensor
receptors and a few taste receptors (Foord et al., 2005). Families 4, 5 and 6 are additional minor
classes that are considerably smaller (Davies et al., 2007).
1.1.1 G Proteins
Agonist stimulated GPCRs undergo conformational rearrangements that allow their
activation of G proteins which leads to modulation of different intracellular effectors. The
classical model of signal transduction cascades mediated by GPCRs involves the binding of an
extracellular ligand to the receptor binding pocket. This leads to a conformational change of the
receptor and allows activation of heterotrimeric G proteins. Heterotrimeric G proteins consist of
one Gα, Gβ and Gγ subunit. There are 27 Gα subunits, 5 Gβ subunits, and 14 Gγ subunits in
total. Receptor activation catalyzes the exchange of guanosine diphosphate (GDP) for guanosine
triphosphate (GTP) on the guanine nucleotide binding/GTPase domain of inactive Gα subunits.
This results in the dissociation of the Gα-GTP and Gβγ subunits, which can then activate or
inhibit various intracellular effectors or enzymes, such as adenylyl cyclases, guanylyl cyclases,
phospholipases and ion channels (Wess, 1998).
Four main Gα families have been described that are based on the primary sequence
similarity of the Gα unit. These include Gαs, Gαi, Gαq and Gα12. The Gαs family contains both
Gαs and Gαolf proteins. The enzyme adenylyl cyclase is activated by Gαs protein subtypes,
resulting in an increased production of cAMP by the enzyme. Gαolf proteins have specific
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expression in the olfactory epithelia and striatum. The Gi family includes Gαi, Gαo, Gαt and
Gαz proteins. Adenylyl cyclase production is inhibited by the activation of Gαi/o protein
subtypes. Except for Gαz, members of the Gαi family are pertussis toxin (PTX) sensitive. Gαi
proteins are inactivated by PTX through ADP-ribosylation, which prevents Gαi proteins from
coupling to the GPCR (Milligan and Kostenis, 2006). The Gαq family consists of Gαq, Gα11,
Gα14, Gα15, and Gα16. All Gαq proteins are activators of phospholipase C-β (PLC) resulting
in production of the second messengers, inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol.
The Gα12 family consists of Gα12 and Gα13 proteins and regulate a group of Rho guanine
nucleotide exchange factors (RhoGEFs). While full characterization of Gα12/13 still remains
incomplete, accumulating evidence indicates that Gα12/13-mediated signaling pathways are
involved in a variety of physiological processes, including embryonic development, cell growth,
cell polarity and migration, angiogenesis, platelet activation, the immune response, apoptosis,
and neuronal responses (Suzuki et al., 2009).
There are 5 Gβ and 14 Gγ subunits in total. The Gβ and γ subunits form a functional unit
that can only be dissociated by denaturing conditions. Distinct combinations of Gβγ subunits
result in specific signaling activity and there are currently a number of proteins that either
interact with or are regulated by Gβγ subunits. Many of the processes dependent on Gβγ
downstream signaling are sensitive to PTX, which selectively modifies the Gαi proteins
(Smrcka, 2008). For example, Gβγ subunits released from Gαi heterotrimers can activate PLCβ.
Although most Gβγ-dependent signaling appears to arise from Gαi proteins, there are a few
examples of PTX-insensitive processes reported to be mediated by Gβγ subunits, as suggested
for the M3 muscarinic Gq coupled receptor (Stehno-Bittel et al., 1995).
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1.2 Control of GPCR Signaling
GPCR signaling is also regulated at the receptor level by distinct processes that include
desensitization, internalization and resensitization. Desensitization is an adaptive process used
by cells to wane membrane signaling and internalization refers to the removal of GPCRs from
the cell surface. The receptors can then recycle back to the plasma membrane allowing for
resensitization of the signal or be retained in intracellular compartments or be targeted for
degradation. These processes are central to the continued maintenance or resultant termination
of the receptor mediated signal (reviewed by Ferguson, 2001).
Several other mechanisms are also used to regulate GPCR signaling. Blocking the
function of presynaptic neurotransmitter transporters that serve to remove endogenous ligands
from the extracellular space surrounding a GPCR increases receptor activation. Additionally,
extracellular ligands are degraded by enzymes, such as acetylcholinesterases to control
transmitter levels (Appleyard, 1994). Furthermore, termination of GPCR signaling can occur at
the level of the G protein by interacting with Regulator of G Proteins Signaling (RGS) proteins,
which increase the rate of hydrolysis of GTP that is bound to Gα subunits and therefore
decrease signaling (reviewed by Hollinger and Hepler, 2002 and Ross and Thomas, 2001).
1.2.1 Agonist-dependent GPCR Desensitization
Activation of a GPCR by its agonist can initiate a process known as desensitization, an
adaptive process used by cells to wane membrane signaling and avoid potentially harmful effects
that can result from excessive cell stimulation. There are two types of desensitization,
homologous and heterologous. Homologous desensitization is an agonist dependent process, with
only the activated receptors desensitized. Heterologous desensitization refers to the activation of
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one receptor leading to a decreased response of an unrelated, unactivated receptor. In either type,
there is functional uncoupling of the receptor from heterotrimeric G proteins in response to
receptor phosphorylation at serine and threonine residues on intracellular domains and the
carboxyl terminal domain (reviewed by Ferguson, 2001). A schematic of the classical regulation
of GPCR signaling is presented in Figure 1-1. GPCR phosphorylation is mediated by both second
messenger dependent kinases, such as cAMP-dependent protein kinase (PKA) and calcium
activated protein kinase C (PKC), and second messenger independent kinases, such as G protein-
coupled receptor kinases (GRKs), casein kinases I and II and tyrosine kinases (Bohm et al.,
1997). Second messenger activated protein kinase phosphorylation of GPCRs is generally
involved in heterologous desensitization and phosphorylation by GRKs is involved in
homologous desensitization (Bunemann et al., 1999).
There are seven known GRK subtypes based on sequence and functional similarity
(Sterne-Marr and Benovic, 1995). These kinases phosphorylate agonist occupied GPCRs, thus
mediating homologous desensitization. GRK2, GRK3, GRK5, and GRK6 account for the
regulation of most GPCRs throughout the body (Gainetdinov et al., 2004). All GRKs contain an
amino terminal RGS-like domain, a central protein kinase domain, and a variable carboxyl
terminal pleckstrin homology (PH) domain (Figure 1-2). Some GRKS also induce receptor
desensitization independent of receptor phosphorylation. For example, the RGS domains of both
GRK2 and GRK3 have been demonstrated to interact with Gq proteins, and therefore
sequestering them and making them unavailable for further signaling (Carman et al., 1999).
Additionally, the carboxyl terminal domain of GRK2 binds to Gβγ subunits to sequester these
proteins (Tobin, 2002). GRKs can also serve as adaptor proteins that facilitate receptor
internalization by interacting with endocytic machinery components such as clathrin (Mangmool
et al., 2006).
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Figure 1-1: Schematic diagram of the classical regulation of GPCR signaling. (1) Desensitization of GPCRs results from the binding of arrestins to agonist occupied receptors following phosphorylation of the receptor by GRKs. Arrestin binding leads to receptor and heterotrimeric G protein uncoupling resulting in termination of signaling by effectors. Arrestins also act as adaptor proteins, binding to components of the clathrin endocytic machinery including clathrin and adaptin, AP-2. (2) GPCR sequestration occurs via dynamin and clathrin coated pits. Once internalized, the receptor exhibits either a Class A or Class B pattern of arrestin interaction and trafficking. Class A GPCRs, rapidly dissociate from arrestin upon internalization. These receptors are trafficked to an acidified endosomal compartment, where the ligand is dissociated and the receptor dephosphorylated by a GPCR-specific protein phosphatase, PP2A, and are subsequently recycled to the plasma membrane (3). Class B GPCRs, form stable receptor-arrestin complexes. These receptors accumulate in endocytic vesicles and are either targeted for degradation or slowly recycled to the membrane (Reproduced with permission from (Luttrell and Lefkowitz, 2002)).
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1.2.2 Agonist-dependent GPCR Internalization
Upon agonist stimulation, GPCRs can also be removed from the cell surface into specific
intracellular compartments through the process of internalization. This process decreases the
number of GPCRs from the cell surface and therefore limits their interaction with agonists in the
extracellular space. The prototypical and most recognized agonist induced internalization
pathway of GPCRs is through clathrin coated pits. This pathway involves the binding of a group
of intracellular proteins known as arrestins to a GRK phosphorylated receptor. This interaction
results in uncoupling of the receptor from its G protein and facilitates GPCR mobilization in
clathrin-coated pits by functioning as a proximal endocytic adaptor molecule to recruit clathrin
and adaptin protein, AP2 (Gainetdinov et al., 2004) (Figure 1-1). The GTPase dynamin then
pinches off the clathrin coated vesicle (Damke et al., 1994). Arrestin association is driven by
recognition of GRK phosphorylation sites on the receptor and the active conformation of the
receptor (Gainetdinov et al., 2004). The arrestins include two visual arrestins (rod and cone)
which are associated with rhodopsin in the retina and arrestin2 and arrestin3 which are
ubiquitously expressed.
Several studies have shown that there are differences in the translocation kinetics of
arrestin2 and arrestin3 to GPCRs, resulting in two classes (A and B) based on their internalization
properties (Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). For Class A receptors,
such as the β2 adrenergic receptor and the dopamine D1 receptor, arrestin3 translocates to the
receptor more readily than arrestin2. The arrestin dissociates from the receptor at or near the
plasma membrane and does not co-internalize with the receptor, allowing for dephosphorylation
and rapid recycling of the receptor back to the cell surface. In contrast, Class B receptors such as
AT1a angiotensin receptor and V2 vasopressin receptor, do not display a preference for either
arrestin isoform.
8
Figure 1-2: General structure of a GRK protein. GRKs consist of an amino terminal RGS region (~185 amino acids), a central protein kinase catalytic domain (~270 amino acids), and a variable carboxyl-terminal domain (~105-230 amino acids). Some of the specific interactions elucidated include GPCRs and Gq with the GRK2 RGS domain and Gβγ with the GRK2 carboxyl terminal PH domain. (Adapted with permission from (Pao and Benovic, 2002)).
9
These receptors form a stable complex with arrestin and traffic together into endocytic
compartments resulting in inefficient dephosphorylation and much slower recycling of the
receptor (Figure 1-1). Specific clusters of serine and thereonine residues in the carboxyl tail have
been demonstrated to be the molecular determinant that defines these internalization properties as
demonstrated by studies using chimeric GPCRs in which this region is exchanged between Class
A and B receptors (Oakley et al., 2001).
1.2.3 GPCR Recycling and Degradation
Once GPCRs are internalized, they may either enter the recycling pathway or degradation
pathway. In the recycling pathway the internalized receptors are dephosphorylated by a GPCR-
specific phosphatase and recycled back to the cell surface to prevent prolonged desensitization
and allow for the resensitization of the signal. In contrast, the receptors can also be targeted to
lysosomes where they are proteolytically degraded, resulting in receptor downregulation. These
two opposing processes are regulated by discrete cellular mechanisms that include the recognition
of specific sorting motifs within the carboxyl tail, as shown for the protease-activated receptor 1
(PAR1) (Trejo and Coughlin, 1999). Selective interactions of the cytoplasmic tail of GPCRs with
sorting proteins, such as sorting nexin 1 (SNX-1), ezrin-radixin-moesin-binding phosphoprotein
(EBP50) and G protein-coupled receptor association protein (GASP) have also demonstrated
importance in post endocytic sorting of GPCRs (Cao et al., 1999; Wang et al., 2002; Whistler et
al., 2002). Lysosomal targeting of GPCRs can also occur by the posttranslational attachment of
ubiquitin, as demonstrated for the yeast α factor mating receptor (Terrell et al., 1998) and the
human CXCR4 chemokine receptor (Marchese and Benovic, 2001).
10
1.3 Introduction to GPCR Oligomerization
Although GPCRs were traditionally believed to exist and function as single monomeric
entities, it is now widely accepted that GPCRs exist as higher-order molecular forms such as
dimers and larger oligomers (George et al., 2002). Oligomerization was shown to occur not only
between the same receptors, forming homooligomers of which a homodimer is the smallest unit,
but also between different receptors, forming heterooligomers. Heterooligomers can not only
form within GPCR families, as was shown for the serotonin (Xie et al., 1999) and opioid families
(Fan et al., 2005; George et al., 2000), but also between unrelated members of different families
as observed between the somatostatin and dopamine receptors (Rocheville et al., 2000) and the
adenosine and dopamine receptor (Gines et al., 2000), to name a few.
The existence of receptor-receptor interactions adds a level of complexity to the
regulation of GPCR functions. Homooligomerization of GPCRs can increase the cooperativity
between GPCR binding sites. For example, ligand binding of one receptor may affect the affinity
of a second receptor within the oligomer. This was suggested by studies on the leukotriene B4
receptor, where agonist binding to one receptor induced specific changes in the conformation of
the ligand binding pocket of the other receptor within the biochemically isolated dimer (Mesnier
and Baneres, 2004). Additionally, activation of several receptors within an oligomer by a single
ligand may lead to amplification of the signal (George et al., 2002).
Heterooligomerization between GPCRs can result in novel features such as altered
binding profiles (Ferre et al., 2007; George et al., 2000) unique trafficking pathways (AbdAlla et
al., 2000) and unprecedented effects upon receptor signaling such as second messenger activation
(Gines et al., 2000) or a switch in the signaling pathway (Rozenfeld and Devi, 2007). GPCR
heterooligomerization may also be relevant to disease states as has been shown for the
11
Angiotensin AT1 and Bradykinin B2 receptors. In this case, altered heterooligomerization
between these two receptors was postulated to be involved in the pathogenesis of angiotensin II
hypersensitivity in preeclampsia (AbdAlla et al., 2001). Additionally, heterodimerization between
the B2-adrenoreceptor and prostaglandin receptors resulted in a reduced bronchodilator response
to B2-adrenoreceptor agonists and therefore was suggested have an impact on asthma (McGraw
et al., 2006). A dysregulation of metabotropic glutatmate 2 and serotonin 5HT2A receptor
heteromer expression in cortical tissue was suggested to result in abnormal signaling that could
predispose schizophrenic patients to psychosis (Gonzalez-Maeso et al., 2008). Furthermore, an
increased expression of dopamine D1-D3 receptor heteromers was hypothesized to be involved in
L-dopa induced dyskinesia in patients with Parkinson’s disease (Fiorentini et al., 2008;
Marcellino et al., 2008b).
1.3.1 Evidence for GPCR Oligomerization
Although the concept of oligomerization has only become widely accepted as a vital
characteristic of GPCRs in the last 10 years, there were earlier observations from radiation
inactivation, chemical crosslinking, and radioligand binding studies that GPCRs might be
organized as oligomers (Bouvier, 2001). The discovery that the GABABR1 and GABABR2
receptors formed functional heterooligomers that allowed for proper GABABR1 transport and
GABABR2 function in 1998 (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998) was
instrumental in making the concept of GPCR oligomerization widely accepted.
Today there are a number of techniques that are used to provide evidence for GPCR
oligomerization, including Bioluminescence Resonance Energy Transfer (BRET), Fluorescence
Resonance Energy Transfer (FRET), co-immunoprecipitation and functional complementation.
12
One important finding that demonstrated very convincing evidence for the existence of GPCR
oligomers was through the use of atomic force microscopy (AFM). AFM is a technique used to
study surface morphology and the properties of molecules, often at atomic resolution. Through
AFM, rhodopsin, the prototypic GPCR of many family 1 GPCRs, was shown to exist on mouse
retinal disc membranes as dimers arranged in rows as oligomers (Fotiadis et al., 2003), suggesting
that other family 1 receptors may have the ability to also orient themselves in a similar
arrangement. Another key technique that has proven useful in supporting the concept of GPCR
oligomerization is x-ray crystallography, which results in high resolution structural images.
Recently, the chemokine CXCR4 receptor was crystallized revealing a structure in favor of
oligomer formation as a similar dimer interface was observed in multiple crystal forms (Wu et al.,
2010). Additionally, the crystal structure demonstrated that binding by a ligand induced a
conformational change within the homoooligomeric receptor complex, providing structural
evidence in support of cooperative binding observed by some ligands.
1.3.2 GPCR Stoichiometry
Although it is known that GPCRs can form receptor-receptor interactions, the actual
proportion of receptors that make up the functional unit of a GPCR complex is still unclear. Most
methodologies do not distinguish if the GPCR exists in a dimer or higher-order oligomeric
complex and there is still controversy about the issue of whether a receptor is functional in a
monomeric form. Some earlier immunoprecipitation studies performed in both heterologous cells
and native brain tissue revealed the presence of monomers, dimers and oligomers, but these
results can also be influenced by the experimental conditions employed, such as denaturing of
receptors due to solubilization with detergents. Monomeric forms have been shown to be present
13
for some GPCRs including rhodopsin receptor (Jastrzebska et al., 2004), neurokinin-1 receptor
(Meyer et al., 2006), N-formyl peptide receptor (Gripentrog et al., 2003), muscarinic M2 (Park
and Wells, 2003), and somatostatin receptors SSTR1 and SSTR5 (Patel et al., 2002). However, as
mentioned previously, the experimental conditions employed in these studies can result in
disruption of interactions mediating oligomerization, challenging the physiological relevance.
More recent studies using energy transfer and fluorescence complementation studies actually
postulate assemblies of GPCRs into larger oligomeric complexes. This has been proposed for
dopamine D2 receptors (Fotiadis et al., 2006; Guo et al., 2008), GABAB receptors (Maurel et al.,
2008), adenosine A2 receptors (Vidi et al., 2008), B2-adrenoreceptors (Dorsch et al., 2009; Fung
et al., 2009) and serotonin 5HT1A receptors (Ganguly et al., 2011).
Furthermore, despite the widely accepted notion that GPCRs exist as dimers or
oligomers, some recent studies of monomeric GPCRs reconstituted in high density lipoprotein-
like particles demonstrate that the monomeric form is sufficient for functional interactions with G
proteins or arrestins. This efficient activation of G proteins was shown for the β2 adrenergic
receptor (Whorton et al., 2007) and rhodopsin receptor (Whorton et al., 2008), with monomeric
rhodopsin also being capable of binding to arrestin (Tsukamoto et al., 2010). Yet, although these
studies indicate a monomer can function in a reconstituted particle, the physiological relevance
for the existence of functional GPCRs as monomers in their native environment is still
questionable. Indeed, rhodopsin has been observed to exist as higher order oligomers in vivo
using AFM (Fotiadis et al., 2003). Furthermore, it is known that for other non GPCR systems,
monomers are not functional and oligomerization is required. This has been demonstrated for
tyrosine kinases (Lemmon et al., 1994), cytokine receptors (Constantinescu et al., 2001), nuclear
steroid receptors (Whitfield et al., 1999), ion channels (Papazian, 1999) and N-methyl-D-aspartic
14
acid (NMDA) receptors (Wenthold et al., 2003). Thus, this requirement for oligomerization may
hold true for GPCRs as well.
1.3.3 Structural Features of GPCR Oligomers
GPCR oligomerization occurs through direct interactions between receptors that are
mediated by both covalent and non covalent bonds, including disulphide, hydrophobic and
electrostatic interactions (Kumar and Nussinov, 2002). These interactions have been suggested to
be mediated by several structural regions of a GPCR, including extracellular, intracellular and
TMs.
The importance of the amino terminal domain in GPCR homooligomerization varies
depending on the family. The involvement of this domain has been revealed for the family 3
metabotropic glutamate 1 receptors based on X-ray crystallographical data of its extracellular
binding region, (Kunishima et al., 2000). Cysteine residues localized on the amino terminal
domain formed a disulphide linked homodimer both in its resting and ligand occupied state.
Disulphide bonds in the amino terminal domain of the calcium sensing receptor were also shown
to be critical for intermolecular receptor interactions (Ray et al., 1999). A role for the amino
terminus in receptor interactions has also been implicated for family 1 GPCRs, such as the
bradykinin B2 receptor, as shown by using mutant bradykinin B2 receptors lacking the amino
terminal domain or peptides corresponding to the amino terminus to block interactions (AbdAlla
et al., 1999).
The carboxyl terminal domain has also been shown to mediate interactions in GPCR
homooligomers and heterooligomers. The carboxyl terminal domain was suggested to be
15
important in the homooligomerization of δ-opioid receptors (Trapaidze et al., 1996), and
cannabinoid CB1 receptors (Wager-Miller et al., 2002). For GPCR heterooligomers, the most
detailed evidence for the carboxyl terminal domain comes from studies on the family 3
GABABR1-GABABR2 receptor heterooligomer. These studies demonstrated that heteromer
formation required an assembly of coil-coiled alpha helices contained in the carboxyl terminal
domain (Margeta-Mitrovic et al., 2000). Carboxyl terminal interactions were also shown to
mediate interactions between the family 1 adenosine A2A-dopamine D2 receptor
heterooligomers, where the carboxyl terminal domain of the A2A receptor interacted with the
third intracellular loop of the D2 receptor (Canals et al., 2004).
Interactions involving different TM’s have also been suggested for GPCR
homooligomers and heterooliogmers. There are several examples that demonstrate the
significance of TM’s in mediating interactions between GPCR homooligomers. The importance
of contact between TM 4 and 5 between rhodopsin monomers to form dimers was demonstrated
as well as contacts between TM 1 and 2 of one rhodopsin dimer connecting to TM 5 and 6 of
another rhodopsin dimer leading to the formation of dimeric rows (Liang et al., 2003).
Endogenous cysteine residues located in TM 3 and 4 have also been shown to play a role in
homodimerization of the serotonin 5HT4 receptor (Berthouze et al., 2005). Additinally, TM 4 was
shown to play a role in the homodimerization of serotonin 5HT2C receptors (Mancia et al., 2008)
and α1β adrenoreceptors (Carrillo et al., 2004; Lopez-Gimenez et al., 2007). Furthermore, for the
dopamine D2 receptor, TM 4 interactions were found to form part of the homo dimerization
interface (Guo et al., 2003; Lee et al., 2003), as well TM 6 and 7 (Ng et al., 1996). For GPCR
heterooligomers, TM 4 was reported to mediate interactions between serotonin 5HT2A-
metabotropic glutamate 2 receptor heterooligomers (Gonzalez-Maeso et al., 2008) and
16
corticotropin releasing hormone- arginine vasotocin VT2 receptor heterooligomers (Mikhailova et
al., 2008).
In 2000, two different models were proposed to describe how TM interactions can
mediate GPCR oligomerization, which were the domain swapping and contact model. In the
domain swapping model, TM’s of different GPCRs are swapped so that there is a reciprocal
exchange of receptor domains. In contrast, the contact dimer model suggests GPCRs interact
laterally to form dimers (Gouldson et al., 2000). Although the domain swapping model has shown
validity for some GPCRs, such as the muscarinic M3-adrenergic α2 heteromer (Maggio et al.,
1993), more recent studies provide evidence that is more in favor of the contact dimer model,
such as the demonstration of rhodopsin monomers organized into two dimensional arrays of
dimers (Fotiadis et al., 2003; Liang et al., 2003).
1.4 Regulation of GPCR Oligomers
1.4.1 Agonist-dependent Desensitization of GPCR Oligomers
GPCR homooligomerization has been demonstrated to be important in recruiting
scaffolding proteins, such as arrestins, for desensitization. For example, the muscarinic M3
receptor homooligomer was shown to recruit arrestin2 only when there was co-expression of wild
type M3 receptors (Novi et al., 2005). When the constituent receptors within the M3 receptor
homooligomer included the wild type M3 receptor and a mutated M3 receptor, there was no
arrestin2 recruitment upon agonist activation. Homooligomerization also allowed for cross
phosphorylation of a nonfunctional chemokine CCR5 receptor that homooligomerized with a
17
functional CCR5 receptor (Huttenrauch et al., 2005), suggesting homooligomerization can
function to amplify desensitization.
GPCR heterooligomerization has also been reported to result in altered desensitization
properties. For example, formation of the somatostatin SSTR2A and SSTR3 heterooligomer
resulted in slower agonist induced desensitization compared to what was observed for their
respective homooligomers (Pfeiffer et al., 2001). Similar to homooligomerization, GPCR
heterooligomerization also allowed for cross phosphorylation of the CCR5 receptor by a GRK
dependent mechanism upon agonist activation of the C5a receptor within a chemokine C5a-CCR5
receptor heteromeric complex (Huttenrauch et al., 2005). Formation of μ-opioid-somatostatin
SST2A receptor heterooligomers, adenosine A2A-dopamine D2 receptor heterooligomers, and μ-
opioid- chemokine CCR5 receptor heterooligomers, also each led to a cross-desensitization of
receptor function (Chen et al., 2004; Hillion et al., 2002; Pfeiffer et al., 2002).
1.4.2 Agonist-dependent Internalization of GPCR Oligomers
GPCR homooligomers have been shown to internalize as a homooligomeric complex. For
example, it was shown that agonist occupancy of only one monomer within the β2-adrenoceptor
homodimer was sufficient to cause co-internalization of each constituent receptor, indicating the
activated β2-adrenoceptor can internalize as a dimeric complex (Sartania et al., 2007).
Similar to homooligomerization, many GPCR heterooligomers have been shown to
internalize as a complex after activation of either member within the complex. This has been
demonstrated for the adenosine A2A-dopamine D2, somatostatin SSTTR2a-μ-opioid, and
α1Β adrenergic -α1D adrenergic receptor heterooligomers (Hague et al., 2004; Hillion et al.,
18
2002; Pfeiffer et al., 2002). It is suggested that activation of either member within the heteromeric
complex can recruit the necessary endocytic machinery, for receptor internalization. GPCR
heterooligomerization can also affect the endocytic properties of the receptors. Indeed, GPCR
heterooligomerization has been demonstrated to both inhibit and induce receptor internalization,
depending on the constituent receptors that form the receptor heterooligomer. For example,
although the β1 adrenergic receptor does not significantly internalize and the β2 adrenergic
receptor significantly internalizes upon agonist activation, heterooligomer formation of the two of
these receptors led to inhibition of agonist induced endocytosis of the β2 adrenergic receptor
(Lavoie et al., 2002). Likewise, heterooligomer formation of the κ-opioid receptor with either the
δ opioid-receptor or β2 adrenergic receptor resulted in inhibition of internalization of these
receptors (Jordan and Devi, 1999; Jordan et al., 2001). In contrast, it has also been shown that
receptors that could not internalize as homooligomers were able to internalize as a result of
heterooligomer formation. This was demonstrated for the somatostatin SSTR1 receptor, which
did not internalize as a homooligomer, but did endocytose when in a heterooligomer formation
with the SSTR5 receptor (Rocheville et al., 2000).
In addition to altering the extent of internalization, heterooligomerization has also been
shown to change the endocytic pathway used by receptor homooligomers after agonist activation.
This was demonstrated for the angiotensin II AT1-bradykinin 2 receptor heterooligomer that
internalized in a dynamin dependent manner even though each constituent homooligomer
endocytosed independently of dynamin (AbdAlla et al., 2000).
GPCR heterooligomerization can also alter the complement of scaffolding proteins, such
as arrestins, that are recruited during endocytosis and may explain some of the changes in
endocytic properties that are observed upon heteromer formation. For example, although the
thyrotropin releasing hormone TRHR2 recruited arrestin3 when expressed as a homooligomer, it
19
recruited arrestin2 when it formed a heterooligomer with the TRHR1 receptor and thus changed
the rate of receptor internalization (Hanyaloglu et al., 2002).
1.4.3 Post Endocytic Sorting of GPCR Oligomers
Homooligomerization of GPCRs has been shown to affect membrane trafficking of the
receptors after agonist induced endocytosis. This was demonstrated for the endocytosed β2
adrenergic receptor that was sorted to a degradative fate as opposed to recycling upon
homooligomerization with a recycling defective mutant version of the β2 adrenergic receptor that
was directed to lysosomes (Cao et al., 2005).
Heterooligomerization of GPCRs has also been demonstrated to change the endocytic
fate of a receptor. Activation of the chemokine CXCR4-chemokine CXCR5 heterooligomer by a
CXCR5 ligand not only resulted in co-internalization of the receptors, but was also suggested to
change the trafficking of the co-internalized CXCR4 receptor from its degradative pathway to the
CXCR5’s recycling pathway (Contento et al., 2008).
As mentioned earlier, GPCRs can be designated as Class A or Class B GPCRs,
depending on the pattern of arrestin recruitment and post endocytic sorting. However, this
designation of Class A or B has been shown to change for GPCRs upon heterooligomerization.
For example, this has been demonstrated for the vasopressin V1a-vasopressin V2a receptor
heterooligomer (Terrillon et al., 2004). When expressed as homooligomers, the V1a receptor
behaves as a class A receptor with arrestin dissociation from the receptor and rapid recycling, and
the V2a receptor is designated as class B, without arrestin dissociation from the receptor and
retainment in endosomes. Yet, upon V1a-V2a receptor heterooligomer formation, co-activation of
20
both receptors resulted in recruitment of the complex with arrestin into endocytic compartments
that did not enable V1a recycling. In contrast, activation of the V1a receptor alone recruited the
heteromeric complex into endosomes without arrestin and resulted in rapid recycling of the
heterooligomer (Terrillon et al., 2004).
1.4.4 Stability of GPCR Oligomers
Even though there is much evidence in support of GPCRs assembling into dimers,
tetramers, or even higher order oligomers, the stability of these complexes is still not known.
Earlier studies which indicate that dimer assembly may occur during biosynthesis and travel to
the cell surface in a dimeric form, suggest a stable and static interaction between the constituent
receptors of the GPCR oligomer (reviewed by Lohse, 2010). However, more recent reports
actually suggest that GPCR oligomers may behave in a more transient and dynamic state, where
the associated receptors also dissociate into monomers in seconds or less. This was suggested for
both the B1-adrenergic receptor (Dorsch et al., 2009) and D2 dopamine receptors (Fonseca and
Lambert, 2009), as shown by fluorescence recovery after photo bleaching (FRAP). FRAP
experiments use receptors that are tagged with fluorophores and an area of the cell membrane is
bleached; this is followed by monitoring the subsequent return of fluorescence into the bleached
area. In the reported studies, a fraction of receptors on the cell surface was immobilized by
crosslinking with an antibody, and the mobility of the non-crosslinked receptors was monitored
by using FRAP. Immobilization of receptors that are part of stable dimers or oligomers also
immobilize associated receptors that are not directly crosslinked. Based on these reports, it has
been proposed that GPCRs may exist in a transient monomer-dimer equilibrium state that may
shift as GPCRs move through various cellular compartments that they encounter from synthesis
21
to degradation (reviewed by Lambert, 2010). Overall, the dynamics of GPCR assemblies of
monomers into dimers or higher order oligomers is still not definitely known and is major topic of
current research.
1.5 Dopamine
The catecholamine neurotransmitter dopamine has fundamental roles in regulating a wide
variety of functions such as locomotion, cognition, reward and emotion. Dopamine also regulates
prolactin release from the pituitary gland, modulates sensory perception in the retina and
olfactory bulb, as well as controls body temperature and food intake (Callier et al., 2003). At the
peripheral level, it controls renal function, gastrointestinal motility, and blood pressure (Missale
et al., 1998). Dysregulation of this system in brain has been implicated in a number of
pathological conditions such as schizophrenia, drug addiction, attention deficit hyperactivity
disorder and Parkinson’s disease (Pivonello et al., 2007).
Dopaminergic neurons synthesize and release dopamine through two reactions. First, the
aromatic amino acid, tyrosine, is converted into L-3,4 dihydroxyphenylalanine (L-Dopa) by the
enzyme, tyrosine hydroxylase (rate limiting step) and then L-Dopa is decarboxylated by aromatic
L-amino acid decarboxylase to produce dopamine (Vallone et al., 2000). As a typical
neurotransmitter, dopamine is released into the synapse after stimulation by an action potential
via a calcium dependent mechanism. In the synaptic cleft, dopamine transmits the signal by
binding to dopamine receptors on pre and post synaptic sites. The dopamine signal is terminated
by the process of reuptake by a presynaptic Na+/Cl- dependent transporter that removes
dopamine from the extracellular space soon after its release, thus regulating its concentration.
After reuptake, dopamine can then be re-packaged for synaptic release. Alternatively it can be
22
inactivated by its metabolic enzymes monoamine oxidase and catechol-O-methyltransferase
(Elsworth and Roth, 1997).
1.5.1 Dopamine Pathways and Functions
There are four main dopamine-mediated pathways in the brain that include the
nigrostriatal, mesocortical, mesolimbic and tuberoinfundibular neurons. The nigrostriatal
pathway originates in the dopamine-synthesizing neurons of the substantia nigra compacta and
extends to the striatum (caudate putamen). This pathway is primarily responsible for the control
of movement. Pathological conditions such as Parkinson’s disease demonstrate the importance of
dopamine in the control of movements. This disease is characterized by a degeneration of
dopaminergic neurons in the substantia niagra resulting in a reduction of circulating dopamine in
the striatum leading to motor impairment. In Parkinson’s disease all components of the
nigrostiatal dopamine pathway degenerate and therefore the dopamine loss is accompanied by a
significant reduction in other neurochemical markers of presynaptic dopamine neurons,
including the dopamine metabolite homovanillic acid as well as the synthesizing enzymes L-
tyrosine hydroxlase and L-dopa decarboxylase. The degree of striatal dopamine loss is correlated
with the severity of the motor symptoms of Parkinson’s disease (Lang and Lozano, 1998).
The mesolimbic pathway innervates the ventral striatum (nucleus accumbens) from the
ventral tegmental area (VTA) and is involved in modulating motivated behaviour. This system
has been implicated in reward mechanisms and psychomotor effects generated by drugs of abuse,
such as cocaine and amphetamine. Administration of psychostimulants and drugs of abuse elicit
an increase in dopamine release in the mesolimbic areas, whereas withdrawal of these drugs
results in a reduction in dopaminergic transmission. In intracranial self-stimulation experiments
performed in rats, the rewarding properties of stimulation results in dopamine release in the
23
prefrontal cortex and nucleus accumbens (Di Chiara and Imperato, 1988; Jackson and Westlind-
Danielsson, 1994).
The mesocortical pathway also arises from the VTA, but extends to various regions of the
frontal cortex, where it is involved with aspects of learning and memory. Dopamine transmission
in this system has been demonstrated to play a role in transient changes of impulse activity in
motivational and attention processes that are essential to learning and cognitive behaviour
(Schultz et al., 1993).
Lastly, the tuberoinfundibular pathway arises from the arcuate nucleus of the
hypothalamus and terminates in the median eminence of the hypothalamus. This pathway is
responsible for transporting dopamine to the anterior pituitary gland (Vallone et al., 2000). This
dopaminergic system results in inhibition of prolactin release. Prolactin is produced in the
lactotrophs of the anterior pituitary gland and plays a significant role in lactation. Loss of
dopaminergic control or drugs with anti-dopaminergic activity can result in over production of
prolactin leading to suppression of secretion hormones such as LH and FSH by inhibiting GnRH
release (Doppler, 1994).
1.6 Dopamine Receptors
The dopamine receptors are members of the GPCR super family and mediate the effects
of dopamine. There are five dopamine receptors, D1, D2, D3, D4 and D5 which are divided into
two distinct subclasses: the D1-like receptors, of which the D1 and D5 are members, and the D2-
like receptors, of which the D2, D3 and D4 receptors, are members. The D1 and D5 receptors
share a high similarity in overall sequence homology (80% with the TMs and 50% overall) and
the D2, D3 and D4 receptors share a high sequence homology with each other (46% for D2 and
24
D3; 53% for D2 and D4 (O'Dowd, 1993)). Two alternatively spliced transcripts are generated
from the D2 receptor gene and code for the D2L (long) and D2S isoforms (short). The D2L
isoform differs from the D2S by the insertion of 29 amino acids in the putative third intracellular
loop of the receptor (Neve et al., 2004). The D1 and D2-like receptors differ in their structure and
their ability to link to second messenger systems. Schematic diagrams of D1 and D2 receptor are
presented in figures 1-3 and 1-4.
1.7 Dopamine D1 Receptors
1.7.1 Cellular Signaling of D1 Receptors
The major signaling pathway of the D1 receptor is stimulation of adenylyl cyclase via
activation of Gs and Golf proteins while the D2 receptor primarily signals through Gi or Go
proteins to inhibit adenylyl cyclase. The overall amino acid homology for human D1 and D2
receptors is 29% but within the TMs, the two receptors are 44% identical (O'Dowd, 1993). The
D1-like receptors have a relatively short third intracellular loop and a long carboxyl terminal
domain while D2-like receptors have a relatively large third intracellular loop and short carboxyl
terminal domain. The cAMP pathway is the most well known signaling pathway associated with
the D1 receptor. D1 receptor adenylyl cyclase activation resulting in cAMP accumulation was
observed in cells (Le Crom et al., 2004) and adenylyl cyclase V was determined to be the isoform
of the adenylyl cyclases that was preferably activated by D1 receptors in brain (Lee et al., 2002).
To activate adenylyl cyclase, the D1 receptor couples to Gs (Pedersen et al., 1994) and Golf
proteins (Zhuang et al., 2000). The D1 receptor was also observed to couple to Gq/11 proteins in
the striatum, hippocampus and amygdala (Jin et al., 2001). Furthermore, the D1 receptors have
also been shown to activate ion channels, PLC and various kinases in addition to adenylyl
cyclases (Gerfen et al., 2002; Maurice et al., 2001; Jin et al., 2001).
25
Figure 1-3: Schematic diagram of the dopamine D1 receptor. The human dopamine D1 receptor consists of 446 amino acids and has a relatively short third intracellular loop (IC3) and long carboxyl tail.
Amino Terminal Domain
Extracellular
IntracellularICL1
ICL3
ICL2
Carboxyl Terminal Domain
Amino Terminal Domain
Extracellular
IntracellularICL1
ICL3
ICL2
Carboxyl Terminal Domain
26
Figure 1-4: Schematic diagram of the dopamine D2 receptor. The human D2 receptor has a relatively large ICL3 and short carboxyl terminal domain. The red residues on the ICL3 illustrate the 29 amino acid difference between the long and short isoforms of the D2 receptor. The short isoform is a 414 amino acid protein and the long isoform is a 443 amino acid protein.
Carboxyl Terminal Domain
ICL2
Extracellular
ICL1
ICL3
Amino Terminal Domain
27
1.7.2 Desensitization of D1 Receptors
Desensitization of D1 receptors has been extensively studied over the past several years
and indicates that dopamine-induced attenuation of signaling by these receptors occurs within
minutes of exposure (Gardner et al., 2001; Jackson et al., 2002; Lamey et al., 2002; Mason et al.,
2002; Ng et al., 1994; Ng et al., 1995; Ng et al., 1997). As with the majority of GPCRs, the
predominant form of D1 receptor desensitization has been identified as being mediated through
GRKs. Truncated mutant constructs of the rat D1 receptor have shown that multiple residues
located downstream of Gly379 in the distal carboxyl terminus regulated dopamine-mediated
phosphorylation and desensitization of the D1 receptor, which was suggested to reflect the
removal of potential GRK2 and/or GRK3 phosphorylation sites (Jackson et al., 2002). Carboxyl
terminal sequences located upstream of Gly379 (between Cys351 and Gly379) were shown to be
important for phosphorylation but not for desensitization (Jackson et al., 2002). Site directed
mutagenesis studies of the human D1 receptor, on the other hand, have provided evidence to
suggest that GRK2 acts as a critical regulator of rapid agonist-induced receptor desensitization
through phosphorylation of a single motif containing the residues Thr360 and Glu359 in the
proximal segment of the carboxyl terminus (Lamey et al., 2002). Both of these studies have used
differential methodologies and different species of the D1 receptor which may play a role in the
discrepant results observed. Site directed mutagenesis studies may be a more reliable method for
identifying the importance of specific residues since there is no change in the intact structure of
the receptor. Carboxyl terminal truncations, however, can alter the structure of the receptor
which permits access to previously sterically hindered receptor domains, such as the third
intracellular loop.
The third cytoplasmic loop has also been implicated in desensitization of the D1 receptor.
It was previously demonstrated that the mutation of specific residues in the third intracellular
28
loop did not affect desensitization of the D1 receptor (Lamey et al., 2002). However, a
subsequent report has demonstrated that these same residues were involved in D1 receptor
phosphorylation and desensitization (Kim et al., 2004). A possible reason for this discrepancy
may be the use of differential cell lines, where one study used Chinese Hamster Ovary (CHO)
cells and the other Human Embryonic Kidney (HEK) 293 cells. It has been shown that the rate of
agonist-induced desensitization of the D1 receptor in CHO cells occurs more slowly than in other
cell types (Ventura and Sibley, 2000). Thus, it has been postulated that D1 receptor
phosphorylation may be GRK isoform-dependent and these isoforms may be lacking in the CHO
cell line (Kim et al., 2004).
Given the evidence demonstrating the importance of the carboxyl terminus and third
intracellular loop, it has been proposed that D1 receptor phosphorylation takes place in both the
carboxyl terminus and third intracellular loop in a sequential manner, where primary
phosphorylation of the carboxyl terminus is permissive for secondary third intracellular loop
phosphorylation, which then allows for the desensitization response (Kim et al., 2004).
In contrast to D1 receptor phosphorylation by GRK2 and GRK3, GRK4 has been shown
to regulate the constitutive phosphorylation and desensitization of the D1 receptor without
exposure to agonist (Rankin et al., 2006) suggesting that specific GRK isoforms may serve
discrete functions in the regulation of dopamine receptor activity.
PKA may also play a role in homologous desensitization of the D1 receptor. Although it
has been demonstrated that the mutation of a potential D1 receptor PKA phosphorylation site
reduced the rate of agonist-induced desensitization (Jiang and Sibley, 1999), and moreover, that
D1 receptor desensitization was blunted in cells deficient in PKA (Ventura and Sibley, 2000), it
has also been shown that the inhibition of PKA action, either by substitution mutations (Lamey
29
et al., 2002; Mason et al., 2002) or pharmacologically (Mason et al., 2002), appeared to have no
effect on D1 receptor-mediated increases in cAMP. These studies were carried out in different
cell lines and therefore suggest that the involvement of PKA for homologous desensitization of
the D1 receptor may be cell type specific (Gardner et al., 2001).
1.7.3 Internalization of D1 Receptors
The acute administration of dopamine agonists has been demonstrated to induce a robust
internalization response both in cultured cells and neurons (Martin-Negrier et al., 2000; Martin-
Negrier et al., 2006) as well as in vivo (Dumartin et al., 1998). While in the absence of agonist
the D1 receptor remained predominantly on the cell surface, the addition of dopamine induced
rapid internalization of approximately 70% of the receptors, with a half life of less than 5 min
(Vickery and von Zastrow, 1999). However, although endocytosis of the D1 receptor has been
consistently documented in both heterologous expression systems and neuronal cultures, the
underlying mechanisms have shown to be more variable. Although earlier studies have identified
a role for PKA-mediated internalization in cells endogenously expressing the D1 receptor (Bates
et al., 1993), mutagenesis of the PKA sites of the rat dopamine D1 receptor (Jiang and Sibley,
1999), the human D1 receptor (Lamey et al., 2002) and the non-human primate D1 receptor
(Mason et al., 2002) did not affect agonist-induced internalization.
Consistent with the role of GRKs in D1 receptor desensitization, this group of kinases
also appears to play an important role in D1 receptor internalization, although the underlying
mechanisms have yet to be fully elucidated. Receptor mutagenesis has revealed that specific
residues in the distal portion of the carboxyl terminus (Thr446, Thr439, and Ser431) are involved
in GRK2-mediated internalization of the D1 receptor (Lamey et al., 2002). However, D1
30
receptor mutants with carboxyl terminal truncations implied sequences located between Cys351
and Gly379 that are pivotal to receptor internalization, but not desensitization (Jackson et al.,
2002). Although there appear to be discrepancies regarding the relative importance of specific
residues in D1 receptor internalization, the carboxyl terminus seems to be essential in this stage
of the endocytic trafficking pathway. Yet, it has also been postulated that GRK-mediated D1
receptor phosphorylation on the third intracellular loop may be of relevance in promoting
receptor interactions with arrestins (Kim et al., 2004). Specifically, it has been suggested that the
phosphorylation of residues within the carboxyl terminus and third intracellular loop dissociates
the two domains allowing for arrestin to bind to the activated third loop (Kim et al., 2004).
Activation of the heterologously expressed D1 receptor leads to translocation of both arrestins2
and 3 to the cell membrane, with arrestin3 being the more predominant translocated subtype.
Following arrestin membrane localization, the D1 receptor is internalized and arrestin
subsequently dissociates from the receptor at or near the membrane (Kim et al., 2004; Oakley et
al., 2000; Zhang et al., 1999). Similarly, co-localization between endogenous D1 receptors and
arrestins in rat neostriatal neuronal cultures demonstrated that the D1 receptor preferentially
interacts with arrestin3 (Macey et al., 2005).
In addition to arrestins, studies assessing the internalization pathway of D1 receptor
membrane trafficking have demonstrated the involvement of numerous other proteins, including
the scaffolding proteins post synaptic density-95 (PSD-95), clathrin, caveolin-1, and the GTPase
dynamin (Kong et al., 2007; Vickery and von Zastrow, 1999; Zhang et al., 2007). In cultured
cells, the co-expression of PSD-95 with the D1 receptor resulted in a robust internalization of the
receptor in the absence of agonist. Additionally, the abolishment of PSD-95 in mice accentuated
D1 receptor-mediated behavioral responses, suggesting that PSD-95 may also serve an inhibitory
role in the regulation of D1 receptor signaling in vivo (Zhang et al., 2007). Evidence suggests
31
that the facilitation of D1 receptor internalization by PSD-95 is mediated through interactions
with the carboxyl terminus of the D1 receptor, and furthermore is dependent upon the presence
of dynamin (Zhang et al., 2007). As dynamin has been previously shown to be involved in
dopamine-induced clathrin-mediated endocytosis of the D1 receptor (Vickery and von Zastrow,
1999) these findings implicate the clathrin-mediated endocytic pathway in the constitutive
internalization of the D1 receptor.
In addition to clathrin-mediated internalization, it has been shown in cultured cells that
the D1 receptor can be localized to low density caveolin-enriched membrane domains and can
associate with caveolin-1 in rat brain through a specific binding motif found in TM 7 (Kong et
al., 2007). Agonist stimulation of the D1 receptor caused translocation of the D1 receptor into
caveolin-1-enriched membrane fractions, which was determined to be the result of D1 receptor
endocytosis through caveolae. However, unlike the relatively rapid clathrin-dependent
mechanism of internalization in which approximately 70% of activated receptors were
internalized within 5 min (Vickery and von Zastrow, 1999), caveolin-dependent D1 receptor
endocytosis appeared to be kinetically slower, reaching approximately 55% internalization
within 45 min of agonist stimulation (Kong et al., 2007). Palmitoylation of the D1 receptor was
hypothesized to play a role in directing the receptor to the slower caveolae-dependent
internalization pathway as opposed to the accelerated clathrin-dependent endocytosis pathway
since a de-palmitoylated D1 receptor exhibited a significantly greater rate of internalization than
wild-type D1 receptor (Kong et al., 2011). These findings suggest that both clathrin- and
caveolin-mediated processes may play functionally distinct roles in regulating D1 receptor
responsiveness in vivo.
32
1.7.4 Resensitization and Recycling of D1 Receptors
Investigations into the trafficking fate of the D1 receptor after agonist induced
internalization have generally reported that the D1 receptor recycles back to the plasma
membrane (Bartlett et al., 2005; Dumartin et al., 1998; Jackson et al., 2002; Lamey et al., 2002;
Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004; Vickery and von Zastrow, 1999), as
opposed to being targeted to lysosomes for receptor degradation. With the use of
immunohistochemistry or fluorescence microscopy, the D1 receptor expressed in cultured cells
or neurons was demonstrated to recycle back to the plasma membrane after removal of agonist
within approximately 20-30 min (Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004;
Vickery and von Zastrow, 1999). In accordance with these studies, dopamine-stimulated D1
receptor phosphorylation has been shown to be rapidly reversed within 30 min. It was suggested
that internalization was not mandatory for D1 receptor dephosphorylation since pretreatment of
the cells with hypertonic sucrose or concanavalin A did not alter D1 receptor dephosphorylation
after agonist removal (Gardner et al., 2001). Resensitization of the D1 receptor-mediated cAMP
response occurred within 90 min (Thompson and Whistler, 2011). The efficient recycling of the
D1 receptor was reported to require a specific sequence within the proximal portion of the
carboxyl terminus of the receptor (Vargas and Von Zastrow, 2004). This sequence spans amino
acid residues 360-382 of the human D1 receptor and is distinct from those previously identified
as being required for efficient recycling of other GPCRs (Cao et al., 1999; Cong et al., 2001;
Gage et al., 2001; Kishi et al., 2001; Tanowitz and von Zastrow, 2003). The importance of this
sequence as a sorting signal was further established by demonstrating that the motif could induce
the recycling of the δ-opioid receptor, a receptor that traffics preferentially to lysosomes after
agonist induced internalization (Vargas and Von Zastrow, 2004).
33
Attempts have been made to elucidate the accessory proteins that may contribute to the
regulation of D1 receptor post-endocytic sorting. GASP has been shown to interact with the D1
receptor, and to a greater degree, the D2 receptor (Bartlett et al., 2005; Thompson et al., 2007).
However, while GASP was demonstrated to promote receptor degradation for the D2 receptor, a
role in D1 receptor sorting was not observed (Bartlett et al., 2005). One study involving a
number of mutant GPCRs including the D1 receptor demonstrated that the presence of a GASP
interaction in and of itself is not sufficient to induce receptor degradation but rather it is the
robustness of the GASP-receptor interaction that regulates the targeting to lysosomes. Although
deletion of the recycling motif in the D1 receptor prevented recycling, it also was not targeted for
degradation, suggesting preventing recycling does not necessarily promote D1 receptor
degradation unless affinity for sorting proteins such as GASP that mediate degradation is altered
as well (Thompson et al., 2007).
1.7.5 D1 Receptor Homooligomers and Heterooligomers
Similar to most GPCRs, D1 receptors have been demonstrated to exist as homooligomers,
forming complexes with identical receptors. An appropriate D1 receptor homooligomeric
conformation must exist for proper signaling and cell surface trafficking (George et al., 1998;
Kong et al., 2006). D1 receptors can also form heterooligomers with other dopamine receptor
subtypes. The D1 receptor heterooligomerized with the D5 receptor but the physiological
significance of this interaction was not determined (O'Dowd et al., 2005). D1 receptors formed
heterooligomers with the D3 receptor with activation resulting in increased affinity of dopamine
for the D1 receptor and potency of dopamine in stimulating adenylyl cyclase through the D1
receptor (Fiorentini et al., 2008). Activation of the D1-D3 receptor heteromer by a D1 agonist
34
also abolished agonist induced D1 receptor internalization, but paired stimulation of the D1-D3
receptor complex by both a D1 and D3 receptor agonist enabled co-internalization of the D1-D3
receptor complex, even though D3 receptors are not reported to significantly internalize when
expressed alone (Kim et al., 2001). Additionally, stimulation of the D1-D3 heteromer by a D3
agonist potentiated D1 receptor mediated behavioral effects (Marcellino et al., 2008b).
Furthermore, D1 receptors can form heterooligomers with D2 receptors in cells and in vivo
resulting in a Gq/11 protein-linked PLC mediated intracellular calcium signal that was not
activated by either constituent receptor alone (Hasbi et al., 2009; Lee et al., 2004; Rashid et al.,
2007). The D1-D2 receptor heterooligomer will be discussed in more detail below.
Formation of D1 receptor heteromeric complexes with other GPCRs have also been
reported, including the adenosine A1 receptor (Gines et al., 2000), ghrelin receptor (Jiang et al.,
2006), μ-opioid receptor (Juhasz et al., 2008), and histamine H3 receptor (Ferrada et al., 2009).
Formation of a D1-A1 receptor heterooligomer resulted in a decreased D1 receptor affinity for
dopamine (Gines et al., 2000) and formation of a D1-ghrelin receptor heteromer resulted in a
switch of G protein selectivity for the ghrelin receptor from Gq/11 to Gi/o proteins with activation
leading to increased D1 receptor mediated cAMP signaling (Jiang et al., 2006). Activation of D1-
H3 receptor heteromers by H3 receptor agonists led to activation of the mitogen-activated protein
kinase (MAPK) signaling cascade, an effect that was not seen in cells expressing H3 receptors
alone (Ferrada et al., 2009) and formation of D1- μ-opioid receptor heterooligomers resulted in a
significantly enhanced surface expression of the μ-opioid receptor (Juhasz et al., 2008).
35
1.8 Dopamine D2 Receptors
1.8.1 Cellular Signaling of D2 Receptors
Similar to the D1 receptor, the cAMP pathway is the most studied pathway associated
with the D2 receptor. However, in contrast to the D1 receptor, the D2 receptor results in
inhibition of adenylyl cyclase and a decrease in cAMP concentrations. The D2 receptor was
found to preferably inhibit adenylyl cyclase V in brain (Lee et al., 2002). To inhibit adenylyl
cyclase, the D2 receptor couples to Gαi and Gαo proteins as well as Gαz proteins (Albert et al.,
1990; Obadiah et al., 1999). D2 receptors are also capable of modulating other second messenger
systems, including inositol phospholipid metabolism, arachidonic acid release, potassium currents
and calcium currents (Banihashemi and Albert, 2002; Memo et al., 1992; Nilsson et al., 1998;
Rasolonjanahary et al., 2002). Additionally, more recent studies indicate that the D2 receptor can
function through the protein kinase B (Akt) – glycogen synthase kinase 3 (GSK-3) signaling
cascade through an arrestin 3 dependent mechanism (Beaulieu et al., 2007; Beaulieu et al., 2005;
Mannoury la Cour et al., 2011). Furthermore, the D2 receptor can affect dopamine mediated
cAMP signaling by forming a complex with the prostate apoptosis response (Par-4) protein (Park
et al., 2005).
1.8.2 Desensitization of D2 Receptors
Early studies examining the functional desensitization of D2 receptors have generated
variable results but, in general, indicate that D2 receptors desensitize much more slowly than D1
receptors and require prolonged agonist treatment (Ng et al., 1997; Zhang et al., 1994). Similar to
the D1 receptor, the mechanisms underlying D2 receptor desensitization appear to involve
36
GRKs. Only by over expression of GRK2, GRK5 (Ito et al., 1999), or GRK3 (Kim et al., 2001)
was there increased phosphorylation of the human D2 receptor and receptor internalization,
indicating the sensitivity of the D2 receptor as a substrate for GRK phosphorylation is lower than
the D1 receptor. A recent study demonstrated that although the D2 receptor is phosphorylated by
GRK2 and GRK3, D2 receptor GRK phosphorylation was not required for agonist induced
receptor desensitization or internalization, but rather was shown to regulate post-endocytic
trafficking of the receptor (Namkung et al., 2009a). Additionally, GRK2 was shown to
constitutively attenuate D2 receptor signaling through a mechanism that required GRK2 kinase
activity and Gβγ binding, but did not involve receptor phosphorylation (Namkung et al., 2009b).
Furthermore, GRK6 may also play a role in regulating desensitization of the D2 receptor. Gene
deletion of GRK6 was shown to lead to enhanced coupling of D2 receptors to their respective G
proteins in vivo, an effect that was associated with increased susceptibility to the locomotor
activating effects of psychostimulants, suggesting that desensitization was inhibited due to
GRK6 knockout (Gainetdinov et al., 2003).
In addition to GRK mediated desensitization, the second messenger kinase, PKC, has
been suggested to be involved in D2 receptor desensitization. PKC phosphorylation was shown
to attenuate the ability of both the D2 receptor to inhibit cAMP accumulation (Cho et al., 2007;
Namkung and Sibley, 2004) and additionally, to induce specific effects on each D2 receptor
isoform (D2L and D2S) with regards to receptor stimulated calcium mobilization (Morris et al.,
2007). It has been reported that although PKC is able to effectively desensitize D2S-induced
increases in intracellular calcium, the D2L isoform is insensitive to PKC-induced desensitization
of calcium signaling due to the presence of a pseudosubstrate domain. A pseudosubstrate domain
is a site that resembles a substrate domain except that the serine phosphorylation site is replaced
by alanine or other residues (Morris et al., 2007). This regulation of substrate sensitivity to PKC
37
appeared to be the result of intramolecular competition between different substrate domains on
the D2L receptor for PKC recognition and a pseudosubstrate domain, which is not found in the
D2S receptor. Given the importance of the D2 receptor in numerous physiological processes, the
presence of pseudosubstrate domains may potentially have significant implications for the
regulation of the receptor by PKC.
1.8.3 Internalization of D2 Receptors
The endocytosis of the D2 receptor is a highly complex process that has been shown to be
both isoform and cell specific, as well as to exhibit both dynamin-dependent and independent
mechanisms (Iwata et al., 1999; Kabbani et al., 2004; Kim et al., 2001; Vickery and von
Zastrow, 1999).
Internalization of the D2 receptor requires increased levels of GRKs and appears to be a
relatively slow process taking approximately 2 hours to plateau (Ito et al., 1999; Itokawa et al.,
1996; Iwata et al., 1999). Whereas little or no internalization was observed in the absence of
exogenous GRKs or in the presence of the dominant negative GRK2, co expression of GRK2,
GRK3 or GRK5 (Ito et al., 1999; Kim et al., 2001), caused significant D2 receptor
internalization. PKC activation also led to 50% of the D2 receptor being internalized when PKC
was over expressed (Namkung and Sibley, 2004). Mutagenesis studies suggest that both of the
PKC phosphorylation domains identified within the third intracellular loop were involved in
regulating its internalization from the cell surface (Namkung and Sibley, 2004).
Similar to the D1 receptor, internalization of the D2 receptor involves translocation of
arrestin2 and arrestin3 to the cell membrane (Kim et al., 2001; Macey et al., 2004) which
38
function to promote receptor internalization (Kim et al., 2004; Namkung et al., 2009a). The
endogenous dopamine D2 receptor in neurons, however, has been shown to preferentially
interact with arrestin2 (Macey et al., 2004). The D2 receptor isoforms also showed differential
regulatory mechanisms for internalization. For example, although both isoforms displayed a
similar level of phosphorylation and arrestin translocation, the actual internalization of the two
isoforms were differentially regulated by GRKs and arrestins, where the internalization of the
D2S receptor was preferentially enhanced by GRK2 or GRK3, but the D2L receptor was
preferentially enhanced by arrestin3 (Cho et al., 2006). As discussed previously, given that the
D2L and D2S receptor isoforms are structurally similar, with the exception of a 29 amino acid
deletion in the third intracellular loop of the D2S receptor, it is plausible that this region may
play a role in isoform specific trafficking.
In contrast to the D1 receptor, D2 receptor internalization appears to be mediated by
specific dynamin isoforms, suggesting specificity between dynamin isoforms and dopamine
receptor subtypes. It has been reported that the internalization of the D2S receptor is dynamin
dependent, implicating the clathrin-coated endocytic pathway in the sequestration of this receptor
(Iwata et al., 1999; Kim et al., 2001; Kim et al., 2004). There are conflicting reports, however, as
to the importance of dynamin-mediated mechanisms in the internalization of the D2L receptor.
While it has been suggested that the D2L receptor internalizes in a dynamin independent manner
(Kim et al., 2004; Vickery and von Zastrow, 1999), these studies assessed only the role of the
dynamin-1 isoform, whereas the dynamin-2 isoform has been more recently implicated. In
cultured cells and primary striatal neurons dynamin-2 was shown to localize to sites of D2
receptor internalization and associate with the D2 receptor in the rat brain (Kabbani et al., 2004).
Furthermore, when an anti-D2 receptor antibody and high-resolution immunoelectron
microscopy was used to study internalization patterns of the D2 receptor in the primate prefrontal
39
cortex, the D2 receptor was demonstrated to undergo clathrin endocytosis via clathrin coated pits
and clathrin coated vesicles (Paspalas et al., 2006).
1.8.4 Resensitization and Recycling of D2 Receptors
Studies examining the trafficking of internalized D2 receptors have generally reported
targetting of the D2 receptors to lysomes for degradation in cells and neurons as opposed to
recycling (Bartlett et al., 2005). As discussed previously, unlike the D1 receptor, the sorting fate
of the D2 receptor appears to be mediated by GASP in non-neuronal cells. Moreover, it was
shown that dopaminergic neurons endogenously expressing GASP did not exhibit a functional
recovery of neuronal responses following D2 receptor agonist administration, whereas disrupting
the GASP- D2 receptor interaction facilitated the recovery of functional D2 receptor responses
(Bartlett et al., 2005). In addition to GASP, the PKC interacting protein, ZIP, has been shown to
associate with the D2 receptor in both cultured cells and endogenous brain tissue. Over
expression of ZIP reduced D2 receptor cell surface expression via enhanced trafficking of the
receptors to lysosomes, suggesting that the ZIP protein functions as a negative modulator of D2
receptor expression (Kim et al., 2008).
1.8.5 D2 Receptor Homooligomers and Heterooligomers
Similar to D1 receptors, D2 receptors exist as homooligomers forming complexes with
identical receptors (Guo et al., 2003; Lee et al., 2000; Lee et al., 2003; Wurch et al., 2001) and
require appropriate homooligomeric formation for proper function (Lee et al., 2000). D2
receptors also form heterooligomers with other dopamine receptor subtypes, including the D3,
40
D1, and D5 receptors. Formation of D2-D3 receptor complexes increased the selectivity of
antiparkinsonian drugs to these receptors (Maggio et al., 2003) as well as resulted in adenylyl
cyclase inhibition by binding of D3 agonists that do not inhibit adenylyl cyclase through D3
receptor activation alone (Scarselli et al., 2001). As mentioned previously, D2 receptors can also
form heterooligomers with D1 receptors in cells and in vivo resulting in a Gq/11 protein-linked
PLC mediated intracellular calcium signal that was not activated by either constituent receptor
alone (Hasbi et al., 2009; Lee et al., 2004; Rashid et al., 2007). The D1-D2 receptor
heterooligomer will be discussed in more detail below. Furthermore, D2 receptors were shown to
form complexes with D5 receptors resulting in a Gq/11 protein linked PLC mediated calcium
signal from both extracellular and intracellular calcium stores (So et al., 2009), unlike the
calcium release triggered by D1-D2 receptor heterooligomers which was only generated from
intracellular stores. Additionally, unlike what is observed for D1 receptors, which activate
significant intracellular calcium mobilization only within a complex with D2 receptors, a robust
calcium signal from both intracellular and extracellular stores was triggered by D5 receptors
expressed alone that was Gq/11 protein linked and PLC mediated. Heterooligomerization with the
D2 receptor led to an attenuation of this D5 receptor mediated signal.
D2 receptors also form heterooligomers with other GPCRs. One of the most documented
includes the dopamine D2-adenosine A2A receptor heterooligomer. A reduction in cAMP
signaling by A2A receptor activation and a decrease in the affinity of D2 agonists for the D2
receptor were mediated by D2-A2A receptor heterooligomers (Kamiya et al., 2003; Kull et al.,
1999). Co-desensitization and co-internalization of both receptors within the heteromeric
receptor complex was observed after prolonged agonist treatment by either A2A receptor or D2
receptor agonists (Hillion et al., 2002). Dopamine D2-cannabinoid CB1 receptor heterooligomers
resulted in the CB1 receptor switching from Gi/o to Gs protein coupling (Jarrahian et al., 2004)
41
and stimulation of CB1 receptors decreased the affinity of D2 receptors for dopamine (Marcellino
et al., 2008a). D2-somatostatin SSTR5 receptor heterooligomer formation potentiated the effects
of dopamine and somatostatin agonists (Rocheville et al., 2000). Agonist-induced
heterodimerization of dopamine D2-somatostatin SSTR2 resulted in increased affinity for
dopamine and enhanced D2 receptor signaling as well as prolonged SSTR2 receptor
internalization (Baragli et al., 2007). Furthermore, dopamine D2-histamine H3 heteromer
formation was suggested to be responsible for an H3 receptor agonist inhibiting and antagonist
potentiating the locomotor activation induced by a D2 agonist (Ferrada et al., 2008).
1.9 The D1-D2 Receptor Heterooligomer
Although D1 and D2 receptors are biochemically and functionally distinct, some
physiological functions require the co-activation of both receptors (Capper-Loup et al., 2002;
Kita et al., 1999; Robertson and Robertson, 1987; Walters et al., 1987). At a mechanistic level
this has been difficult to reconcile since co-activation of the D1 and D2 receptors can result in
both opposing as well as synergistic physiological responses. The discovery, however, of a
common functional output generated by the concurrent activation of D1 and D2 receptors within
the same cells resulting in activation of a novel Gq/11-linked PLC-dependent calcium signal (Lee
et al., 2004) has provided a possible biochemical mechanism by which the D1 and D2 receptors
work in concert to mediate these molecular and behavioral functions. The calcium signal was
inhibited by either a D1 or D2 receptor antagonist, indicating that both constituents of the
heterooligomer must be activated to elicit the signal. This calcium signal was independent of
extracellular calcium influx as well as other intracellular pathways that can trigger calcium
release, as shown by inhibitors of multiple components of these signaling cascades (Hasbi et al.,
42
2009; Lee et al., 2004). Additionally, the rate of the calcium signal propogation was temporally
similar to what has been detected for Gq-coupled P2Y1 purinergic receptors (Lee et al., 2004),
which result in a calcium signal that has a slower upstroke than the P2Y2 isoform of purinergic
receptors (Gallagher and Salter, 2003).
1.9.1 D1-D2 Receptor Heterooligomer in vitro and in vivo
Heterodimerization of D1 and D2 receptors was initially shown by
coimmunoprecipitation from rat and human brain (Lee et al., 2004). These findings were then
confirmed in HEK 293T cultured cells co-expressing both receptors by FRET (So et al., 2005),
co-trafficking studies (So et al., 2005), and visualization of D1-D2 receptor heteromer co-
trafficking in live cells (O'Dowd et al., 2005).
More recently, quantitative FRET in situ has been utilized to verify the presence of D1-
D2 receptor heteromers both in neonatal cultured rat striatal neurons and adult striatum (Hasbi et
al., 2009; Perreault et al., 2010). In neonatal cultured rat striatal neurons, immunocytochemistry
revealed that D1 and D2 receptors were mainly expressed at the cell surface and on proximal
neurites with a high degree of co localization. Localization of D2 receptors was also observed in
the cytosol. Confocal FRET analysis of the natively expressed D1 and D2 receptors
demonstrated a relative distance of 5-7 nm (50-70Å) in localized microdomains, thus indicating a
physical interaction between them. FRET efficiency ranged from 0.1-0.5, with a higher
efficiency in the soma and proximal dendrites and lower in distal processes (Hasbi et al., 2009).
In adult striatum, the highest degree of D1 and D2 receptor co localization was found in the cell
bodies of neurons in the nucleus accumbens and globus pallidus and the lowest incidence in the
caudate putamen. FRET analysis demonstrated that ~91% of the cell bodies in neurons co-
43
expressing D1 and D2 receptors in the nucleus accumbens exhibited heteromer formation
whereas only ~24% of D1 and D2 receptor co expressing neurons in the caudate putamen formed
the D1-D2 receptor complex. Furthermore, these D1-D2 receptor heteromers were also found on
presynaptic terminals in the striatum (Perreault et al., 2010).
1.9.2 Activation of the D1-D2 Receptor Heterooligomer in Striatum
Activation of the D1-D2 receptor heteromer in adult rodent striatum induced activation of
Gq/11, as shown by direct [35S]GTPγS incorporation into Gq (Rashid et al., 2007). Gq activation
did not occur in either D1 or D2 receptor knockout animals, emphasizing the importance for
involvement of both receptors to induce Gq activation. Similar to the heterologous cells,
treatment of rodents with either a D1 or D2 receptor antagonist blocked the effect of agonist
activation, indicating that activation of both D1 and D2 receptor subtypes that were part of the
heterooligomeric complex was necessary for generating a functional response.
Recently, activation of the D1-D2 receptor mediated calcium signal through the Gq/ PLC
signaling pathway was also shown in neonatal cultured rat striatal neurons (Hasbi et al., 2009).
Similar to the heterologous cells, the calcium signal was inhibited by either a D1 or D2 receptor
antagonist, was independent of extracellular calcium influx as well as other intracellular
pathways that can trigger calcium release, as shown by inhibitors of multiple components of
these signaling cascades. Preincubation with inhibitors of the Gq protein, IP3 receptors, or PLC
resulted in significant inhibition of the D1-D2 receptor heteromer mediated calcium signal,
confirming the involvement of the Gq/PLC pathway in these neurons. Furthermore, no
significant difference in the calcium signal was observed in D5 knockout animals, but it was
abolished in D1 knockout animals, confirming that the calcium mobilization was through the D1-
D2 receptor heterooligomer without involvement of the D5 receptor.
44
1.9.3 Functional consequences of D1-D2 Receptor Heterooligomer Mediated Signaling
Activation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) was the first
demonstration of a functional consequence of activation of the D1-D2 receptor heteromer
complex (Rashid et al., 2007). CaMKIIα plays a fundamental role in synaptic plasticity, and both
its translation and activity can be regulated by increases in intracellular calcium (Lisman et al.,
2002). Significant increases in phosphorylated CaMKIIα in neurons of nucleus accumbens shell
and caudate nucleus was demonstrated within 10 min of an intraperitoneally injection of D1 and
D2 agonists concurrently into mice and rats (Rashid et al., 2007). This effect did not occur in D1
or D2 receptor knockout animals and preadministration of a D1 or D2 antagonist also blocked
this effect, indicating the necessity for both D1 and D2 receptors.
D1-D2 receptor heteromer mediated induction of CaMKIIα and nuclear translocation
was also recently demonstrated to occur in cultured postnatal rat striatal neurons (Hasbi et al.,
2009). Within 2 min of agonist treatment, there were not only robust increases in phosphorylated
CaMKIIα levels, but also a significant increase of phosphorylated CaMKIIα in the nucleus. This
effect was blocked by D2 antagonist treatment and was absent in D1 knockout animals, but still
present in D5 knockout animals. Accordingly, levels of brain derived neurotrophic factor
(BDNF), a neurotrophin whose gene expression is modulated by isoforms of CaMKII (Takeuchi
et al., 2000), were also significantly increased within 1 hour of activation of the D1-D2 receptor
heteromer in these neurons and the effect was blocked by D1 and D2 antagonist treatments, but
still present in D5 knockout animals. Increased BDNF expression was also observed in the adult
rat striatum, specifically in the nucleus accumbens, after activation of the D1-D2 receptor
heteromer. Furthermore, activation of the D1-D2 receptor heteromer by dopamine agonists led to
accelerated morphological maturation and differentiation of the cultured striatal neurons. These
45
results are consistent with the ability of dopamine and BDNF to promote neuronal maturation,
differentiation, and survival (Iwakura et al., 2008). Thus, taken together, this data suggests that
activation of the D1-D2 receptor heteromer signaling pathway is a key cascade involved in the
development, maturation, and differentiation of striatal neurons, through mobilization of
intracellular calcium through Gq, activation of PLC, followed by activation of CaMKIIα and
BDNF signaling.
Alterations in striatal D1-D2 receptor heteromeric function have also been demonstrated
in response to amphetamine (AMPH) treatment (Perreault et al., 2010). It was shown that
repeated AMPH treatment in rats significantly increased the proportion of the D1-D2 receptor
heteromer in the agonist detected high affinity state in striatum and also increased the affinity of
the dopamine agonist for the D1-D2 receptor complex by approximately 10-fold, as shown by
radioligand binding assays. The striatal D1-D2 heteromer high affinity state was absent in D1
knockout animals, but still present in D5 knockout animals. AMPH treatment also resulted in
increased sensitivity of striatal D1-D2 receptor heteromer G protein activation by dopamine, as
shown by agonist induced GTPγS binding, indicating the D1-D2 receptor heteromer was
functionally supersensitive in response to repeated increases in dopamine transmission.
Furthermore, changes in D1-D2 receptor heteromer function was also studied in human
schizophrenia brain samples. Radioligand binding studies demonstrated that there was an
approximate 10-fold increase in affinity of the D2 receptor within the D1-D2 receptor heteromer
for agonist in schizophrenia brain globus pallidus, together with a concomitant increase in the
levels of heteromeric D2 receptors in the high affinity state compared to that in normal
individuals (Perreault et al., 2010). Moreover, there was enhanced D1 and D2 coupling in
postmortem brain of subjects diagnosed with major depression and disruption of the complex
resulted in anti-depressant like effects in rats (Pei et al., 2010).
46
Behavioral consequences of D1-D2 receptor heteromer signaling have also been
examined (Perreault et al., 2010). Selective activation of the D1-D2 receptor heteromer by SKF
83959 significantly increased the amount of time rats spent grooming, an effect that was greatly
attenuated by acute injection of the D2 receptor antagonist, raclopride. In contrast, SKF 83822, a
drug that activates only D1 receptor homooligomers, attenuated grooming.
1.9.4 Pharmacology of the D1-D2 Receptor Heterooligomer
Initially, the pharmacology of the D1-D2 receptor heteromer was studied by investigating
any differences in ligand binding affinity for the D1-D2 receptor heteromer vs. D1 and D2
receptor homooligomers. Results from radioligand binding studies with dopamine and other
commonly used D1 and D2 dopamine agonists, such as SKF 81297 and quinpirole, demonstrated
no differences between the ligand binding pocket of the D1 or D2 receptors within the D1-D2
receptor heterooligomer compared to that within their respective homooligomers (Lee et al.,
2004; So et al., 2005). These unchanged binding characteristics were also similar for D1 and D2
receptor selective antagonists.
However, further studies on the pharmacology of the D1-D2 receptor heteromer revealed
pharmacological profiles with differential specificity for the D1-D2 receptor heteromer and their
respective homomers (Rashid et al., 2007). Radioligand binding assays demonstrated that the
commonly used D1 agonist, SKF 81297, displayed a high affinity for the D1 receptor in D1-D2
receptor heteromers and in D1 receptor homooligomers. This agonist demonstrated robust
activation of the D1 receptor homooligomers through the Gs-mediated adenylyl cyclase pathway
as well as activation of the D1-D2 receptor heteromer through the Gq-mediated PLC pathway.
Two other commonly used D1 agonists that were screened include SKF 83959 and SKF 83822,
both of which displayed a very high affinity for the D1 receptor in D1-D2 receptor
47
heterooligomers and in D1 receptor homooligomers, but displayed differential functional effects.
SKF 83959 was shown to selectively activate the D1-D2 receptor heteromer resulting in a robust
PLC dependent rise in intracellular calcium, without activating D1 receptor homooligomers
coupled to Gs mediated adenylyl cyclase activity. In contrast, SKF 83822 was shown to
selectively activate D1 receptor homooligomer-Gs mediated adenylyl cyclase activity, without
activating the D1-D2 receptor heteromer-Gq mediated calcium signal (Figure 1-5).
Furthermore, results from radioligand binding assays revealed that although SKF 81297
and SKF 83959 are full D1 agonists, they are also partial D2 agonists for the D2 receptor within
the D1-D2 receptor complex (Rashid et al., 2007). This partial D2 receptor agonism was first
suspected when these SKF compounds were shown to generate a calcium signal on their own
with an approximately 70% lower peak height than that generated by dopamine in HEK 293T
cells co-expressing D1 and D2 receptors. This signal could be blocked by D1 or D2 receptor
antagonists and was not seen in HEK 293T cells expressing D1 receptors alone. Accordingly,
when these SKF compounds were co-administered with the D2 agonist, quinpirole, the calcium
signal was increased to the same extent as generated by dopamine. The calcium signal was not
activated by the administration of quinpirole alone in HEK 293T cells co-expressing D1 and D2
receptors or in HEK 293T cells expressing the D2 receptor alone. Thus, these results suggested
that these compounds could directly activate the D2 receptor within the D1-D2 receptor
complex. This was confirmed with radioligand binding assays using PTX to uncouple Gi/o
proteins from D2 receptor homomers leaving them with low affinity for agonists. The results
indicated that SKF 81297 and SKF 83959 could bind to a high affinity PTX-resistant D2 site
within the D1-D2 receptor heteromer in rat and mouse striatum as well as in HEK 293T cells co-
expressing D1 and D2 receptors. This binding was absent in cells expressing only the D1 or D2
receptor and in D1 knockout animals (Rashid et al., 2007).
48
Figure 1-5: Differential agonist activation of the D1-D2 receptor heterooligomer and D1 receptor homooligomer. SKF 81297 activates both the D1-D2 receptor heteromer and D1 receptor homooligomer, while SKF 83959 selectively activates D1-D2 receptor heterooligomer-Gq mediated signaling and SKF 83822 only activates D1 receptor homooligomer-Gs mediated signaling.
SKF 81297SKF 83959
SKF 81297SKF 83822
SKF 81297SKF 83959
SKF 81297SKF 83822
49
1.9.5 D1-D2 Receptor Heterooligomer Desensitization
Although little is known regarding the regulation of D1-D2 heterooligomer
responsiveness, it has been shown that desensitization of the agonist-induced calcium signal
occurs within minutes of agonist exposure and is initiated by agonist occupancy of either
receptor subtype, even though the signal is generated only by occupancy of both receptors (So et
al., 2007). Additionally, the attenuation of receptor internalization did not result in a concomitant
decrease in the magnitude of the desensitization, suggesting desensitization of the signal
occurred prior to recruitment of the complex into vesicles by endocytic machinery. Although
GRKs 5 or 6 or any of the second messenger kinases such as PKA, PKC, CaMKII, or casein
kinases I and II did not play a role in the desensitization, GRKs 2 and 3 appeared to have a role
in the extent of desensitization. Inhibition of GRK2-mediated phosphorylation, however, did not
inhibit this desensitization (So et al., 2007), suggesting that in addition to phosphorylating
receptors, GRKs may also mediate signal desensitization by phosphorylation independent
mechanisms. It has been suggested that GRK2 and GRK3 may sequester Gq/11 proteins, which
interact with the RGS domain on these GRKs (Iwata et al., 2005). Thus, this may provide a
mechanism by which GRKs 2 and 3 contribute to desensitization of the calcium signal mediated
by the D1-D2 receptor heterooligomer (So et al., 2007). It is of note, however, that
heterooligomeric D1 and D2 receptors exhibit conformational changes that permitted cross
phosphorylation of the D2 receptor by selective D1 receptor activation (So et al., 2005), a finding
that implicates a discrete mechanism by which the D1 receptor within the D1- D2 complex may
regulate heterooligomer functioning.
50
1.9.6 D1-D2 Receptor Heterooligomer Internalization
Given the relatively recent discovery of the D1-D2 receptor heteromer, there is much as
yet unknown regarding the regulation and trafficking of this complex. It has been determined
that selective agonist occupancy by either a D1 agonist or D2 agonist leads to D1-D2 receptor
heteromer co-internalization (So et al., 2005). This interesting finding indicates that activation of
only one receptor within the D1-D2 complex is sufficient for internalization, whereas co-
activation of the D1 and D2 receptors is required for the PLC-mediated calcium signal. It was
also shown that heteromerization resulted in altered steady-state cellular distribution of the D1
and D2 receptors within HEK 293T cells that was distinct from that of the D1 and D2 receptor
homomers (So et al., 2005). Together, these findings emphasize the unique trafficking responses
of the heteromer compared to its constituent D1 and D2 receptors, a characteristic that may
elucidate differences in physiological function.
1.10 Research Rationale and Objectives
Since it is well documented that activation of the D1-D2 receptor heteromeric complex
results in calcium signaling in vivo, it is important then to determine how this signal is regulated.
For dopamine receptors, regulatory processes, such as desensitization, may be relevant to the
outcome of hyper-dopaminergic states, such as schizophrenia, as well as to the development of
therapeutic tolerance in the treatment of dopamine related diseases. When I started my PhD
studies, although much was known about how signals generated from D1 and D2 receptor
homooligomers are regulated, the regulation of the D1-D2 receptor heteromer was much less
defined. However, it was known that pre-treatment of the D1-D2 receptor heteromer with
dopamine resulted in rapid desensitization of the D1-D2 receptor mediated calcium signal that
51
occurred at the receptor level and was independent of intracellular calcium store depletion,
suggesting that the calcium signal desensitization might occur at the level of the receptor
complex. Additionally, several second messenger kinases were tested as potential mediators of
D1-D2 receptor heteromer calcium signal desensitization, but only GRK 2 or 3 were shown to
play a role.
Furthermore, when I began my PhD, the pharmacology of the D1-D2 receptor heteromer
had just been thoroughly investigated with selective dopaminergic agonists. It was specifically
identified that the agonist, SKF 83959, selectively activated the D1-D2 receptor heteromer, while
SKF 83822 only activated the D1 homooligomer and SKF 81297 activated both the D1 and D1-
D2 receptor complexes.
Given this knowledge that the D1-D2 receptor heteromer could be selectively activated, I
was able to use the dopaminergic agonists, all of which have equivalent ability to bind with high
affinity to the D1 receptor, but exhibit differential abilities to activate the D1-D2 receptor
heteromeric pathway as pharmacological tools to help elucidate the regulatory characteristics of
the D1-D2 receptor heterooligomer and distinguish them from D1 and D2 receptor
homooligomeric units. Investigating the regulatory characteristics of the D1-D2 receptor
heterooligomer was the overall goal of the project.
Hypothesis: Given that co-activation of the dopamine D1-D2 receptor heterooligomer results in a
rapid and robust Gq-mediated calcium signal, the regulation of this complex will include
mechanisms that are distinct from that regulating its constituent receptors which are not coupled
to Gq.
52
The specific hypotheses were:
1) The D1-D2 receptor complex will desensitize following agonist activation and may
include mechanisms involving mediators such as GRK2.
2) After agonist activation, the D1-D2 receptor heteromeric complex will internalize and
result in resensitization of the calcium signal.
3) Selective agonists will have specific roles in desensitization, internalization and
resensitization of the D1-D2 receptor heteromeric complex and its associated calcium signal.
The objectives of the project were to:
1) Determine desensitization properties of the D1-D2 receptor heterooligomer and identify
mechanisms involved.
2) Evaluate agonist induced internalization of the D1-D2 receptor heterooligomer and
resensitization of the calcium signal.
53
2 MATERIALS AND METHODS
2.1 Cell Culture - All cell culture and transfection reagents were obtained from Invitrogen
(Carlsbad, CA). HEK293T cells were maintained as monolayer cultures at 37 oC in advanced
minimum essential medium supplemented with 6% fetal bovine serum, 300 μg/ml Zeocin, and
antibiotic-antimycotic. Stable cell lines co-expressing the amino-terminal hemagglutinin (HA)
epitope-tagged human D1 receptor and amino-terminal FLAG epitope-tagged human D2
receptor were created in HEK293T cells using the bicistronic pBudCE 4.1 vector (Invitrogen,
Burlington, ON, Canada). Briefly, the D1 receptor cDNA was inserted into the EF1 α
multicloning site and the D2 receptor cDNA into the CMV site. For stable cell lines expressing
either D1 or D2 receptors, HA-D1 receptor cDNA or FLAG-D2 receptor cDNA was introduced
alone into the pBUDCE4.1 vector. Antibiotic-resistant clones (selected with 300μg/ml zeocin) of
each transfection were isolated and tested for expression of corresponding receptors using
saturation binding analysis. All experiments were performed with the long isoform of the D2
receptor.
2.2 Transient Transfections in HEK293T Cells- Cells were grown to 80% confluency before
being transfected with Lipofectamine 2000 reagent (Invitrogen). Experiments were performed 48
hours post transfection. For studies involving GRKs, transient transfections of cDNA encoding
GRK2, K220R-GRK2, D110A-GRK2, or R106A/K220R-GRK2 in the mammalian expression
vector pcDNA3 or enhanced green fluorescent protein-GRK2 (EGFP-GRK2) in the mammalian
expression vector pEGFP-N were performed. GRK constructs were a kind gift from Dr. Jeffrey
Benovic (Thomas Jefferson University, Philadelphia, PA). For small interfering RNA (siRNA)
silencing of gene expression, chemically synthesized double-stranded siRNA duplexes (with 3’
54
dTdT overhangs) were purchased from Qiagen Inc. (Mississauga, ON, Canada). GRK2 siRNA
(5’-AAGAAGUACGAGAAGCUGAG-3’) and a nonsilencing RNA duplex (5’-
UUCUCCGAACGUGUCACGU-3’) that was used as a control for the siRNA experiments were
transfected with a final concentration of 40 nM. The effect of siRNA transfection was assessed
by immunoblotting. Average percent protein knockdown in these experiments was 69% of basal
levels.
2.3 Measurement of the Calcium Signal in HEK293T Cells- Calcium mobilization assays
were carried out using a Flex station multiwell plate fluorometer (Molecular Devices, Sunnyvale,
CA, USA). Stably transfected HEK 293T cells were seeded in black microtiter plates at a density
of 1.5 x 105 cells/well grown for 24 h. The cells were then loaded with 2 μM Fluo-4
acetoxymethyl ester indicator dye (Invitrogen) in advanced minimum essential medium
supplemented with 2.5 mM probenecid (Sigma Aldrich, Oakville, ON, Canada) and 250 μM
Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) (Sigma) for 1 h. The
cells were then washed twice with Hanks’ balanced salt solution (HBSS) supplemented with 20
mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 250 μM EGTA. Baseline
fluorescence values were measured for 15 sec and changes in fluorescence corresponding to
alterations in intracellular calcium levels upon the addition of agonists thereafter were recorded.
Fluorescence values were collected at 3 sec intervals for 100 sec and the difference between
maximum and minimum fluorescence values for each agonist concentration was determined and
analysed using Prism software (GraphPad, San Diego, CA, USA). For desensitization studies,
cells were pre-treated with agonists in serum-free advanced minimum essential medium in a dose
and time response manner and washed off with HBSS supplemented with 250 μM EGTA before
calcium measurement. For resensitization studies, the cells were incubated at 37 oC for up to 3
55
hrs after agonist wash off before calcium measurement. For inhibitor studies, cells were pre-
treated with 10 μM SQ 22536 1 h before agonist pre-treatment, or 10 μM SCH 23390 or
raclopride 10 min before agonist pre-treatment. SKF 81297, SKF 83959, SKF 83822, dopamine,
quinpirole, SCH 23390, raclopride, ATP, H-89 and SQ 22536 were purchased from Sigma.
2.4 Membrane Preparation and Radioligand Saturation Binding Assay- HEK 293T cells co-
expressing D1 and D2 receptors were first treated with either vehicle or 100 nM SKF 83959 in
advanced minimum essential medium supplemented with 2.5 mM probenecid for 30 min at 4oC.
The cells were then washed twice in cold HBSS with probenecid, collected, and centrifuged at
2000 RPM for 10 min at 4 oC to obtain a pellet. Cell lysates were prepared by disruption with a
polytron homogenizer (Kinematica, Basel, Switzerland) in ice cold lysis buffer (5 mM Tris-HCl
and 2 mM EDTA) containing protease inhibitors (5 μg/ml leupeptin, 10 μg/ml benzamidine, and
5 μg/ml soybean trypsin inhibitor). Lysates were centrifuged at 800 RPM for 10 min and the
supernatant was collected. Membrane fractions were prepared by centrifuging the supernatant at
13000 RPM for 20 min. Membrane protein was determined by the Bradford assay according to
the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Saturation binding assays were
performed in 1ml antagonist binding buffer (50 mM Tris HCl, 5 mM EDTA, 1.5 mM CaCl2, 5
mM MgCl2, 5 mM KCl, 120 mM NaCl) with 35 μg of membrane homogenate and increasing
concentrations (0.05 to 4 nM) of [3H]SCH 23390. Non specific binding was determined by 10
μM (+)-butaclamol. Incubation was performed for 2 h at room temperature. At the end of the
incubation, bound ligand was isolated by rapid filtration through a 48-well cell harvester
(Brandel, Montreal, QC, Canada) using Whatman GF/C filters (Whatman, Clifton, NJ, USA).
56
Data were analyzed by nonlinear regression analysis using GraphPad Prism for the determination
of dissociation constants (Kd) and the density of receptors (Bmax).
2.5 Intact Cell Radioligand Binding Assay- HEK 293T cells expressing D1 receptors, D2
receptors or both D1 and D2 receptors were treated with either vehicle, 100 nM SKF 83959 or 1
μM dopamine for 5 or 30 min in advanced minimum essential medium at 37ºC. The cells were
then washed once in acidic buffer (1mM EDTA, 50mM TrisHCl, pH 5) and twice in warm PBS.
Dissociated cells were collected in cold PBS and centrifuged at 1000g for 10 min at 4oC. The
resultant pellet was gently resuspended in antagonist binding buffer and protein estimation was
determined by the Bradford assay according to the manufacturer’s instructions. 35 μg of protein
was incubated with 1 nM [3H]SCH 23390 (Kd = ~ 0.2 nM) or 2 nM [3H]Raclopride (Kd = ~ 1
nM) to a final volume of 1ml. Non specific binding was determined by 10 μM (+)-butaclamol.
Incubation was performed for 3 h on ice to prevent receptor internalization. At the end of the
incubation, bound ligand was isolated by rapid filtration, as described above. To ensure that
[3H]SCH 23390 or [3H]Raclopride measured cell surface receptors, the assay was conducted at 4
ºC and specific binding to cell surface receptors was calculated by the displacement of
radioligand by 100 μM dopamine. Since dopamine does not cross the cell membrane it would
only displace radioligand from receptors on the cell surface. Non- specific binding accounted for
approximately 5% of total bound ligand.
2.6 Immunocytochemistry of HEK293T Cells- HEK 293T cells stably expressing HA-D1 and
Flag-D2 receptors were grown on glass coverslips in 6 well plates for 24 hr. The cells treated
with 1 μΜ dopamine or SKF 83959 for 30min in advanced minimum essential medium at 37oC
57
and then washed three times with PBS. After removal of agonist, the cells were incubated at
37oC for 0 hr, 1.5 hr or 3 hr before fixing with 4% paraformaldehyde for 30 min at 37oC. The
primary antibodies used were rat anti-HA (Roche, Laval, QC, Canada, 1:500) and mouse anti-
Flag (Sigma, 1:500). The secondary antibodies used were anti-rat conjugated to fluorophore
Alexa Fluor 488 and anti-mouse conjugated to flurophore Alexa Fluor 647 (Invitrogen 1:500).
Paraformaldehyde-fixed cells were incubated with the primary antibodies overnight at 4ºC. After
three washes with PBS-Tween20, the samples were incubated with secondary anit-rat and anti-
mouse antibodies for 2 hours at room temperature. After three washes, the glass coverslips were
mounted on slides using a mounting solution (Dako, Carpinteria, CA, USA) and the images were
acquired using a confocal Fluoview Olympus microscope (FV 1000). All images were acquired
in sequential mode to minimize any bleed-through.
2.7 Neuronal Cultures- Neuronal cultures derived from post natal day 1 rodent striatum were
trypsinized in HBSS with 0.25% trypsin and 0.05% DNase (Sigma) at 37ºC, and then washed 3
times in HBSS with 12 mM MgSO4. Cells were dissociated in Dulbeco’s Modified Eagle
Medium with 2 mM glutamine and 10% FBS and plated at 2x105 cells per poly-L-lysine coated
well (Sigma 50 μg/ml). After 24 h the media was replaced with neurobasal medium with 50X
B27 supplement and 2 mM glutamine (Invitrogen). After three days in culture, cytosine
arabinoside was added (5 μM) to inhibit glial cell proliferation. Half of the medium was changed
every three days. The neurons were in culture for 7-14 days before experiments were performed.
Cell viability was tested with trypan blue (0.4%) (Invitrogen) exclusion and indicated 2% cell
death during this time period.
58
2.8 Immunocytochemistry of Cultured Neurons- Neurons that were grown on glass coverslips
in 24 well plates were washed twice in PBS and then fixed in 4% paraformaldehyde for 30 min
at 37oC. The primary antibodies used were rat anti-D1 (Sigma, 1:400), and rabbit anti-D2
(Millipore, Billerica, Massachusetts, USA, 1:400). The secondary antibodies used were anti-rat
conjugated to fluorophore Alexa Fluor 568 and anti-rabbit conjugated to fluorophore Alexa Fluor
647 (Invitrogen 1:500). Paraformaldehyde-fixed neurons were incubated with the primary
antibodies overnight at 4ºC. After three washes with PBS-Tween20, the samples were incubated
with secondary anti-rat and anti-rabbit antibodies for 2 hours at room temperature. After three
washes, the glass coverslips were mounted on slides using a mounting solution and the images
were acquired using a confocal Fluoview Olympus microscope (FV 1000). All images were
acquired in sequential mode to minimize any bleed-through. For GRK2 studies, the neurons were
pretreated with 100 nM dopamine, SKF 83959, or SKF 83822 for 5 min and then washed twice
with PBS before fixing with paraformaldehyde. The primary antibody used was rabbit anti-
GRK2 (Sigma, 1:200) and the secondary antibody used was anti-rabbit conjugated to
fluorophore Alexa Fluor 350 (Invitrogen 1:500).
2.9 Confocal Microscopy FRET- Paraformaldehyde-fixed striatal neurons from rat brain were
incubated for 24 hours at 4°C with primary antibodies highly specific to D1 and D2 receptors
(Lee et al., 2004), and the species-specific secondary antibodies conjugated to Alexa 568 and
Alexa 647 dyes, respectively. The primary antibodies have been shown to be highly specific to
D1 or D2 receptors using HEK 293T cells expressing individual D1, D2, D3, D4 or D5 receptors
(Lee et al., 2004). They were further validated by immunohistochemistry showing lack of
reactivity in striatum slices from D1-/- and D2-/- mice. Anti-D2-Alexa 350 and anti-D1-Alexa 488
were used as the donor and acceptor dipoles, respectively. An Olympus Fluoview FV 1000 laser
59
scanning confocal microscope with a 60X/1.4 NA objective was used to obtain the images. A
Krypton laser at 405 nm and an Argon laser at 488 nm were used to excite the donor and
acceptor, respectively. The emissions were collected at 430 and 530 nm LP filter. Other FRET
pairs (488-568 and 568-647) were tested and showed comparable results. Each FRET analysis
was performed using eleven images and calculated using an algorithm (Chen, 2005). The
corrected FRET (cFRET) images were then generated based on the described algorithm, in
which: cFRET = UFRET – ASBT – DSBT, where UFRET is uncorrected FRET and ASBT and
DSBT are the acceptor and the donor spectral bleed-through signals. Small regions of interest
(ROI), using the same images and software, were used to estimate the rate of energy transfer
efficiency (E) and the distance (r) between the donor (D) and the acceptor (A) molecules in
accordance with the following equation: Efficiency: E = 1 - IDA / [IDA+ pFRET*((ψdd / ψaa )*
(Qd/Qa))], where IDA is the donor image in the presence of acceptor, ψdd and ψaa are collection
efficiencies in the donor and acceptor channels, Qd and Qa are the quantum yields. E is
proportional to the 6th power of the distance (r) separating the FRET pair. r = Ro [(1/E) – 1]1/6,
Ro is Förster’s distance.
2.10 Measurement of the Calcium Signal in Primary Striatal Neurons- Calcium mobilization
was measured in neonatal neurons in culture using cameleon YC6.1 (Truong, et al., 2001), an
engineered calcium indicator based on the conformational change of a calmodulin (CaM) peptide
flanked by two fluorophores, cyan fluorescent protein (CFP) and yellow fuorescent protein (
YFP). An increase in calcium binding to CaM leads to a decrease in the distance separating the
two flanking proteins, CFP and YFP, and results in a measurable energy transfer. The cameleon
was a generous gift from Dr. M. Ikura, University of Toronto. The striatal neuronal cultures were
transfected with cameleon YC6.1 using a combination of Effectene (Qiagen, Mississauga, ON,
60
Canada) and ExgenTM500 (Fermentas, Burlington, ON, Canada) which resulted in a transfection
efficiency of 40-70%. Briefly, 2.7 μg of cameleon YC.6.1 cDNA was mixed with Buffer EC
(Effectene kit), 150 mM NaCl, and Exgen reagent and enhancer and incubated at room
temperature for 5 min. Subsequently, Effectene reagent was added and mixed. Ten minutes later,
6 ml of culture media was added to the mixture, which was split into 24 wells. Treatment with
trypan blue (0.4%) indicated that cell death was ~ 10% after transfection. The experiments were
then performed in these live neurons 48 hours post transfection with cameleon in the absence of
extracellular calcium. Using a single excitation wavelength at 405 nm, which solely excites CFP,
images and fluorescence emissions data for both CFP and YFP were collected and energy
transfer was calculated (Chen, 2005). Activation of the calcium signal was measured following
treatment with either 100 nM dopamine, SKF 83959, or SKF 83822. The background signal was
subtracted from the values obtained after drug injection. For desensitization studies, the neurons
were pre-treated with agonists in HBSS for 30 min and washed off with the same media before
calcium measurement. For inhibitor studies, the neurons were pretreated with 10 μM SQ22536
or H-89, for 30 min before agonist pretreatment. For siRNA experiments, the neurons were
transfected with either GRK2 siRNA or a non-silencing siRNA in cultured medium using
HiPerFect transfection reagent (Qiagen), as per the manufacturer’s instructions. Briefly, 3 μl
HiPerFect reagent was mixed with siRNA diluted in culture medium without serum and
incubated for 10 min at room temperature. The mixture was then added drop wise onto the
neurons in each well. The siRNA final concentration was 40 nM. Experiments were performed
48 hours post siRNA transfection. The effect of siRNA transfection was assessed by Western
blotting analysis. Average knock down of protein expression in these experiments was 70% of
basal levels.
61
2.11 Immunoprecipitation- HEK 293T cells stably expressing both the HA-D1 receptor and
Flag-D2 receptor were transfected with 2 μg GRK2 cDNA using Lipofectamine 2000. 48 hours
post transfection, the cells were treated with 1 μM dopamine, SKF 83959 or SKF 83822 in
advanced minimum essential medium for 5 min at 37 oC and then washed twice with ice cold
PBS. The cells were collected and centrifuged at 2000 RPM for 10 min at 4oC to obtain a pellet.
Cell lysates were prepared by disruption with a polytron homogenizer (Kinematica, Basel,
Switzerland) in ice cold lysis buffer containing protease inhibitors. Lysates were centrifuged at
800 RPM for 10 min and the supernatant was collected. Membrane fractions were prepared by
centrifuging the supernatant at 13000 RPM for 20 min. The resultant pellet was solubilized with
NP-40 for 2 hrs at 4oC and then centrifuged at 15000g for 20 min. The supernatant was collected
for protein determination by the Bradford assay (BioRad). 500 μg of total cell lysate was used
for immunoprecipitation. After pre clearing for 30 min with 10μl protein G agarose beads
(Sigma), the lysates were incubated overnight with 75 μl of anti-FLAG M2-Agarose (Sigma) at
4ºC to immunoprecipitate the FLAG-D2 receptor. Immuno-complexes were collected and
washed 4 times followed by an overnight incubation with 100 μg of a FLAG peptide at 4ºC to
displace the anti-FLAG antibody from the D2 receptor. The proteins were resolved by gel
electrophoresis. Immunodetection of the FLAG-D2 receptor from immunoprecipitates was
detected with mouse anti-FLAG antibody (1:1000) (Sigma) and HA-D1 receptor and GRK2
immunoreactivity were detected with rat anti-HA antibody (1:1000) (Roche) and rabbit anti-
GRK2 antibody (1:1000) (Sigma), respectively.
62
2.12 BRET Assay- To detect an agonist induced interaction between the D1-D2 receptor
heteromer and GRK2, BRET studies were performed on HEK 293T cells transfected with 1 μg
cDNA of Renilla luciferase-D1 (Rluc-D1) receptor, FLAG-D2 receptor, EGFP-GRK2 or empty
vector EGFP. Cells were seeded into 96 well plates at a density of 105 cells/well for 24 hours and
then treated with 1 μM dopamine, SFK 83959, SKF 83822 in HBSS for 1 to 10 min at 37 ºC.
After the induction of Rluc-mediated light emission by the addition of 5μM of the substrate
coelenterazine h (Discoverx, San Diego, CA, USA) emission was measured using a plate-reader
spectrofluorometer (Victor3, Perkin-Elmer) at wavelengths 480 and 535 nm, corresponding to the
maxima of the emission spectra for Rluc and EGFP, respectively. The Net BRET ratio was
calculated by subtracting the background BRET signal obtained from Rluc-D1 receptor in the
presence of empty vector, EGFP from the BRET signal obtained from Rluc-D1 receptor in the
presence of EGFP-GRK2 as defined by the following calculation: [(emission at 535 nm)/
(emission at 480] – Cf where Cf corresponds to (emission at 535)/(emission at 480) from cells
expressing the Rluc-D1 receptor and EGFP.
2.13 SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting- Harvested HEK 293T
cells were solubilized in lysis buffer + a protease and phosphatase inhibitor, sonicated for ~ 30
sec on ice and protein estimation was determined using the Bradford assay (BioRad). The same
procedure was performed for the neurons except they were solubilized in RIPA buffer (1M Tris,
1M NaCl, Na-Deoxycholate, Igepal CA-630, 0.5M EDTA, 500 mM MgCl2). Samples were
prepared in 2X sample buffer (0.5M Tris HCl, glycerol, 10% SDS, 1% Bromophenol blue, β-
Mercaptoethanol) and boiled for 3 min. Samples were separated on pre-cast 10% polyacrylamide
gels (Invitrogen) for 2 hrs at 128V. Proteins were transferred to polyvinylidene fluoride (PVDF)
membranes (Amersham Biosciences, Piscataway, NJ, USA) at 33 V for 2.5 hrs and blocked for 1
63
hour in 5% skim milk powder and then incubated with the appropriate primary antibody
overnight at 4ºC. Rabbit anti-GRK2 antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA)
was used at 1:1000 dilution to assess overexpression of GRK2 and 1:600 to measure knockdown
of GRK2. Rabbit anti-GAPDH (Abcam, Cambridge, MA, USA) was used at 1:7500 dilution.
Blots were rinsed 3 times with TBS-tween and incubated with horseradish peroxidase-
conjugated goat anti-rabbit secondary antibody (1:4000) (Santa Cruz biotechnology, Santa Cruz,
CA) for 2 hrs at room temperature. Immunoreactivity was detected by enhanced
chemiluminescence (ECL) using an ECL plus kit (Amersham, Biosciences).
2.14 Statistical Analysis- All pharmacological data were analyzed using the computer program
GraphPad Prism version 3.00 for Windows. Saturation binding curves were analyzed by
nonlinear regression analysis for the determination of dissociation constants (Kd) and the density
of receptors (Bmax). Data from multiple experiments were averaged and expressed as the means
± Standard Error of the Mean (SEM). Statistical significance at the p<0.05 level is denoted with
* and was determined using the unpaired Student’s t test or one way ANOVA followed by
Tukeys post hoc test.
64
3 RESULTS
3.1 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal in HEK293T
Cells- To study the effects of the agonists on the D1-D2 receptor heteromer mediated calcium
signal, we first confirmed the abilities of each agonist to activate the intracellular calcium signal
in a HEK 293T stable cell line co-expressing D1 and D2 receptors (Lee et al., 2004) in the
presence of EGTA, an extracellular calcium chelator. The cells expressed D1 and D2 receptors in
a 1:1 ratio with final receptor densities of each that were approximately 0.8 pmol/mg protein.
The addition of either dopamine, SKF 83959, the agonist that selectively triggers
phosphoinositide hydrolysis, or SKF 81297, the agonist which activates both adenylyl cyclase
and phosphoinositide turnover, stimulated a robust calcium signal that peaked within 20-40 sec
of agonist activation and declined within 120 sec, compared to vehicle as shown in a
representative tracing (Fig 3-1A) or the peak heights of the calcium signals (Fig 3-1B). The
addition of SKF 83822, the agonist that has been shown to only activate adenylyl cyclase, did not
elicit a significant calcium signal (Fig 3-1A,B).
3.2 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal in
HEK293T Cells- To investigate calcium signal desensitization, the HEK 293T cells were pre-
treated for 5 min with increasing concentrations of each agonist, from 10-11 to 10-4 M, then was
washed off followed by subsequent activation with 10 µM dopamine. All three agonists by prior
exposure were able to significantly desensitize the calcium signal to dopamine activation (60.1 ±
2.8% reduction for SKF 81297 (n=3), 74.4 ± 2.2% reduction for SKF 83959 (n=5), and 62.8 ±
2.8% reduction for SKF 83822 (n=8)).
65
FIGURE 3-1. Specificity of dopamine receptor agonists activating the D1-D2 receptor heteromer calcium signal in HEK 293T cells stably expressing the D1 and D2 receptors. (A) Representative tracings displaying changes in fluorescence corresponding to changes in intracellular calcium levels on treatment of D1 and D2 receptors with 1μM concentrations of dopamine, SKF 83959, SKF 81297, SKF 83822 or vehicle. A.F.U. = absolute fluorescence units. (B) Peak heights of agonist induced calcium release through activation of the D1-D2 receptor heteromer. Values shown are the means ± S.E.M. of n=3 experiments. A significant difference from vehicle is denoted by * = p < 0.05.
8395
981
297
8382
2
Vehicl
e0
25000
50000
75000
100000
125000
AFU
Dopamine
*
* *
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
B
DrugAddition
8395
981
297
8382
2
Vehicl
e0
25000
50000
75000
100000
125000
AFU
Dopamine
*
* *
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
B
8395
981
297
8382
2
Vehicl
e0
25000
50000
75000
100000
125000
AFU
Dopamine
*
* *
8395
981
297
8382
2
Vehicl
e0
25000
50000
75000
100000
125000
AFU
Dopamine
*
* *
Dopamine
*
* *
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822010000200003000040000
50000AFU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Dopamine
83959
81297
Vehicle
83822
A
B
DrugAddition
66
SKF 83959 was the most potent in desensitizing the signal (EC50 = 29.1 ± 0.5nM), followed by
SKF 83822 (EC50 = 54.7 ± 1.2nM), and the least potent was SKF 81297 (EC50 = 130.9 ± 4.1nM)
(Fig 3-2A-D). The potency of the agonists to induce desensitization and the extent of
desensitization of the calcium signal after agonist exposure for 10 or 30 min was similar to that
seen after agonist exposure for 5 min (Fig 3-3 and 3-4 A-C). The experiments were performed in
the presence of 250 µM EGTA, indicating the calcium signal was from intracellular calcium
stores. To demonstrate that depletion of intracellular calcium stores by prolonged agonist
treatment was not the mediator of this calcium signal desensitization, endogenously expressed
purinergic receptors, which also use the Gq protein and PLC as a means to generate calcium
release through intracellular stores (Schachter et al., 1996), were activated with 10 µM ATP after
dopamine agonist pre-treatment in the presence of EGTA. No significant difference in the extent
of the ATP mediated calcium signal was observed after pre-treatment with SKF 83959 (for 30
min) compared to control as determined by their peak heights, suggesting that calcium stores
were not significantly depleted after dopamine agonist pre-treatment (Fig 3-5). To demonstrate
that the desensitization of the calcium signal was not due to residual agonist persistently
occupying the ligand binding pocket of the receptor, saturation binding studies were carried out
after the HEK 293T cells co-expressing D1 and D2 receptors were treated with 100 nM SKF
83959 for 30 min and then washed off. The Bmax and Kd values for [3H]SCH 23390 binding
were 0.892 ± 0.17 pmol/mg protein and 277 ± 9.6 pM for the control cells not pre-treated with
agonist and 0.875 ± 0.13 pmol/mg protein and 308 ± 11 pM (n=4), for cells pre-treated with SKF
83959. These results indicated that there was no persistent occupancy of the ligand binding
pocket after agonist wash off.
67
FIGURE 3-2. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 5 min. (A) Representative calcium tracings displaying the calcium signal activated by 10 μM dopamine (control) and after pre-treatment with 1μM SKF 83959 for 5 min in HEK 293T cells stably expressing D1 and D2 receptors. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (B), SKF 83959 (C), or SKF 83822 (D), from 10-11 M to 10-4 M for 5 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=3-6 experiments.
01000020000300004000050000A
FU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Pretreatment
Control
83959
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
Log [SKF 81297 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
D
01000020000300004000050000A
FU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Pretreatment
Control
83959
01000020000300004000050000A
FU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Pretreatment
Control
01000020000300004000050000A
FU
Time (seconds)
60000700008000090000
100000
0 20 40 60 80 100
Pretreatment
Control
83959
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
Log [SKF 81297 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
D
68
FIGURE 3-3. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 10 min. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (A), SKF 83959 (B), or SKF 83822 (C), from 10-11 M to 10-4 M for 10 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=3-6 experiments.
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959(M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 81297(M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959(M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 81297(M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
69
FIGURE 3-4. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 30 min. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (A), SKF 83959 (B), or SKF 83822 (C), from 10-11 M to 10-4 M for 30 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=5-8 experiments.
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -20
25
50
75
100
Log [SKF 81297 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -20
25
50
75
100
Log [SKF 81297 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83959 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2-25
0
25
50
75
100
Log [SKF 83822 (M)]
% R
educ
tion
ofPe
ak H
eigh
t
A
B
C
70
FIGURE 3-5. The ATP induced calcium signal is not reduced by prior treatment with the agonist, SKF 83959. There was no significant difference in the extent of the ATP mediated calcium signal after pre-treatment with SKF 83959 for 30 min compared to control as determined by their peak heights. A.F.U. = absolute fluorescence units. Values shown are the means ± S.E.M. of n=3 experiments.
0
50000
100000
150000
SKF 83959 - +
ATP
Indu
ced
AFU
71
Since SKF 83822 was not able to significantly activate the calcium signal, but still led to its
desensitization, the involvement of a heterologous mechanism involving the adenylyl cyclase
pathway was investigated. When cells were pre-treated with SKF 83822 in the absence and
presence of the adenylyl cyclase inhibitor, SQ 22536, there was no significant difference in the
extent of the calcium signal, suggesting the desensitization mediated by SKF 83822 did not
involve adenylyl cyclase (Fig 3-6). Taken together, these results demonstrated that although SKF
83959 and SKF 83822 have differential abilities to activate the D1-D2 receptor heteromer
mediated calcium signal, both were able to elicit significant calcium signal desensitization.
3.2.1 Desensitization through selective occupancy of the D1 receptor- Because SKF 81297
and SKF 83959 act as full agonists for the D1 receptor and partial agonists for the D2 receptor
within the D1-D2 receptor complex (Rashid et al., 2007), the desensitization observed could
potentially be mediated by occupancy of both receptors. To investigate whether both D1 and D2
receptors were involved in the desensitization of the signal, a D2 selective antagonist, raclopride,
10 µM, was added with SKF 81297, SKF 83959 or SKF 83822 pre-treatment for 30 min and
then washed off followed by activation with 10 µM dopamine. No significant difference in the
extent of desensitization caused by any of the agonists was observed in the presence of
raclopride (Fig 3-7A-C). However, the desensitization induced by each drug was abolished by
pre-treatment with the D1 antagonist, SCH 23390, 10 µM. These results suggested that the
desensitization elicited by each agonist occurred through selective occupancy of the D1 receptor.
72
FIGURE 3-6. Effect of the adenylyl cyclase inhibitor, SQ 22536, on the SKF 83822 induced desensitization of the D1-D2 receptor heteromer mediated calcium signal. Data represent the percent reduction in peak calcium levels after pre-treatment with 1 μM SKF 83822 in the absence and presence of SQ 22536 (500 μM). Values shown are the means ± S.E.M. of n=4 experiments.
0
25
50
75
SKF83822SQ 22536
+ +- +
% R
educ
tion
ofPe
ak H
eigh
t
73
FIGURE 3-7. Effect of dopamine receptor antagonists on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. Desensitization of the calcium signal elicited by dopamine following pre-treatment with 1μM SKF 81297 (A), 1μM SKF 83959 (B) or 1μM SKF 83822 (C) for 30 min without or with pre-treatment with 10 μM raclopride or 10 μM SCH 23390 (n=4-6).
0
25
50
75
100
SKF 81297SCH 23390Raclopride
++
+
---
-+
+-
-
-
* *
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
0
25
50
75
100
SKF83959SCH23390Raclopride -
-+ + +
+-+ -
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF83822SCH23390Raclopride
+ + +
-- ++
--
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
A
B
C
0
25
50
75
100
SKF 81297SCH 23390Raclopride
++
+
---
-+
+-
-
-
* *
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
0
25
50
75
100
SKF83959SCH23390Raclopride -
-+ + +
+-+ -
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF83959SCH23390Raclopride -
-+ + +
+-+ -
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF83822SCH23390Raclopride
+ + +
-- ++
--
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF83822SCH23390Raclopride
+ + +
-- ++
--
---
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
A
B
C
74
To further confirm the observed desensitization was through occupancy of the D1 receptor rather
than the D2 receptor, the cells were pre-treated with both a D1 agonist and the D2 agonist,
quinpirole, for 30 min and then washed off followed by a subsequent challenge with dopamine.
No significant difference in the extent of desensitization was observed, suggesting that
occupancy of the D1 and D2 receptors concurrently did not enhance desensitization of the signal
(Fig 3-8A-C). Pre-treatment with quinpirole alone did not induce any significant desensitization.
3.3 Activation of the D1-D2 Receptor Mediated Calcium Signal in Primary Striatal
Neurons- D1 and D2 receptors were mainly expressed at the cell surface and on proximal
neurites with a high degree of colocalization in postnatal striatal neurons as determined by
immunocytochemistry (Fig 3-9A). Localization of D2 receptors was also observed in the cytosol.
Confocal FRET analysis of the natively expressed dopamine D1 and D2 receptors demonstrated
a relative distance of 5-7 nm (50-70Å) in localized membrane microdomains, thus indicating a
physical interaction between the natively expressed D1 and D2 receptors. FRET efficiency (E)
ranged from 0.1-0.5, with a higher efficiency in the soma and proximal dendrites and lower in
distal processes as shown in a representative figure (Fig 3-9A). Cameleon was transfected into
the striatal neurons and was used as an indicator for the calcium signal. The majority of
cameleon was localized in the cell bodies as well as a small amount in the dendritic processes
(Fig 3-9B). In the absence of extracellular calcium, addition of either 100 nM dopamine or SKF
83959 to the striatal neurons led to rapid increases in cameleon FRET, corresponding to a rise in
intracellular calcium, as shown in a representative tracing (Fig 3-9C) or the peak heights of the
calcium signals (Fig 3-9D).
75
FIGURE 3-8. Effect of D2 receptor agonist, quinpirole, on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. Desensitization of the calcium signal elicited by dopamine following pre-treatment with 1 μM SKF 81297 (A), 1 μM SKF 83959 (B), or 1 μM SKF 83822 (C) in the absence and presence of 1 μM quinpirole or quinpirole treatment alone (n=3-4). A significant difference from control is denoted by * = p < 0.05.
0
25
50
75
100
-- -
+ -++ +
SKF 83822Quinpirole
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF 83959Quinpirole
-- -
+ -++ +
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF 81297Quinpirole
--
+-
++ +
-
* *
Dop
amin
e In
duce
d %
Pea
k Fl
uore
senc
e
A
B
C
0
25
50
75
100
-- -
+ -++ +
SKF 83822Quinpirole
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
-- -
+ -++ +
SKF 83822Quinpirole
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF 83959Quinpirole
-- -
+ -++ +
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF 83959Quinpirole
-- -
+ -++ +
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
0
25
50
75
100
SKF 81297Quinpirole
--
+-
++ +
-
* *
Dop
amin
e In
duce
d %
Pea
k Fl
uore
senc
e
A
B
C
76
FIGURE 3-9. Specificity of dopamine receptor agonists activating the D1-D2 receptor heteromer calcium signal in primary striatal neurons. (A) Immunocytochemistry showing endogenously expressed dopamine D1 and D2 receptor colocalization (merged) and interaction (corrected FRET = FRETc). The inset shows the calibration for FRET efficiency. (B) Striatal neurons displaying the presence of transfected cameleon (blue). (C) Representative tracings of cameleon FRET resulting from intracellular calcium release from D1-D2 receptor heteromer activation by 100 nM dopamine or SKF 83959 but not SKF 83822. The arrow indicates the time point of drug addition. (D) Peak heights of agonist induced cameleon FRET corresponding to calcium release through activation of the D1-D2 receptor heteromer. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from SKF 83822 is denoted by * = p < 0.05.
0 10 20 30 40 50 60 70-0.050.000.050.100.150.200.25
Time (Seconds)
NET
FR
ET
Dopamine
83959
83822
D1 R D2 R
MERGED FRETc
A B
C D
Dopamine
SKF 8395
9
SKF 8382
20.00
0.05
0.10
0.15
0.20
0.25
NET
FR
ET
27cells
20cells
32cells
*
*
0 10 20 30 40 50 60 70-0.050.000.050.100.150.200.25
Time (Seconds)
NET
FR
ET
Dopamine
83959
83822
0 10 20 30 40 50 60 70-0.050.000.050.100.150.200.25
Time (Seconds)
NET
FR
ET
Dopamine
83959
83822
D1 R D2 R
MERGED FRETc
D1 R D2 R
MERGED FRETc
A B
C D
Dopamine
SKF 8395
9
SKF 8382
20.00
0.05
0.10
0.15
0.20
0.25
NET
FR
ET
27cells
20cells
32cells
*
*
Dopamine
SKF 8395
9
SKF 8382
20.00
0.05
0.10
0.15
0.20
0.25
NET
FR
ET
27cells
20cells
32cells
*
*
77
The FRET signal peaked within 10 sec of agonist activation and declined within 50 sec. In
contrast, induction of calcium mobilization by SKF 83822 was minimal (Fig 3-9C,D).
3.4 Desensitization of the D1-D2 Receptor Mediated Calcium Signal in Primary Striatal
Neurons- For desensitization studies, the neurons were pre-treated with 100 nM SKF 83959 or
SKF 83822 for 30 min and then washed off, followed by subsequent activation with 100 nM
dopamine. Pre-treatment with either SKF 83959 or SKF 83822 led to a significant attenuation of
the calcium signal peak heights (50.9 ± 0 .01% of control for SKF 83959 and 48.8 ± 0.01% of
control for SKF 83822) (Fig 3-10A). The extent of desensitization was not significantly different
when the neurons were pre-treated with SKF 83822 in the presence or absence of either the
adenylyl cyclase inhibitor, SQ 22536, or the protein kinase A (PKA) inhibitor, H-89, suggesting
a heterologous mechanism involving the adenylyl cyclase or cAMP pathway was not responsible
for the signal attenuation by SKF 83822 (Fig 3-10B). Pre-treatment with either inhibitor alone
did not result in any significant difference in the calcium signal from that elicited by dopamine.
Since it was shown that SKF 83959 can occupy both the D1 and D2 receptors within the
D1-D2 receptor complex (Rashid et al., 2007), the desensitization observed may be mediated by
occupancy of both receptors. To prevent agonist occupancy of the D2 receptor, a D2 selective
antagonist, raclopride 10µM was added with SKF 83959 or SKF 83822 pre-treatment for 30 min
and then washed off followed by activation with 100 nM dopamine. No significant difference in
the extent of desensitization with either SKF 83959 or SKF 83822 was observed in the presence
of raclopride (Fig 3-11A and B). However, SKF 83959 and SKF 83822 mediated desensitization
was abolished by pre-treatment with the D1 antagonist, SCH 23390 10 µM.
78
FIGURE 3-10. The D1-D2 receptor heteromer mediated calcium signal is desensitized in striatal neurons by prior treatment with dopamine agonists for 30 min. (A) Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83959 or 100 nM SKF 83822 for 30 min. (B) Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83822 without and with 10 μΜ SQ 22536 or 10 μΜ H-89, or SQ 22536 alone or H-89 alone. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from control (no pre-treatment) is denoted by * = p < 0.05.
B
A
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959SKF 83822
--
+-
-+
Dop
amin
e In
duce
d N
et F
RET
38cells
52cells 49cells* *
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822SQ 22536H-89
+ + +- -+- - +
---
--
-
++-
Dop
amin
e In
duce
dN
et F
RET
**47cells
45cells 57cells
41cells 30cells 47cellsB
A
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959SKF 83822
--
+-
-+
Dop
amin
e In
duce
d N
et F
RET
38cells
52cells 49cells* *
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959SKF 83822
--
+-
-+
Dop
amin
e In
duce
d N
et F
RET
38cells
52cells 49cells
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959SKF 83822
--
+-
-+
Dop
amin
e In
duce
d N
et F
RET
38cells
52cells 49cells* *
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822SQ 22536H-89
+ + +- -+- - +
---
--
-
++-
Dop
amin
e In
duce
dN
et F
RET
**47cells
45cells 57cells
41cells 30cells 47cells
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822SQ 22536H-89
+ + +- -+- - +
---
--
-
++-
Dop
amin
e In
duce
dN
et F
RET
**47cells
45cells 57cells
41cells 30cells 47cells
79
FIGURE 3-11. Effect of dopamine receptor antagonists on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in striatal neurons. Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83959 (A) or 100 nM SKF 83822 (B) without and with 10 μM raclopride or 10 μM SCH 23390. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from control (no pre-treatment) is denoted by * = p < 0.05.
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822RacloprideSCH 23390
+ + +
- +- -+
-
---
Dop
amin
e In
duce
dN
et F
RET
* *
46cells
50cells 50cells
49cells
A B
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959RacloprideSCH 23390
+ + ++- -
- - +
---
Dop
amin
e In
duce
dN
et F
RET
46cells
* *48cells 53cells
33cells
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822RacloprideSCH 23390
+ + +
- +- -+
-
---
Dop
amin
e In
duce
dN
et F
RET
* *
46cells
50cells 50cells
49cells
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83822RacloprideSCH 23390
+ + +
- +- -+
-
---
Dop
amin
e In
duce
dN
et F
RET
* *
46cells
50cells 50cells
49cells
A B
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959RacloprideSCH 23390
+ + ++- -
- - +
---
Dop
amin
e In
duce
dN
et F
RET
46cells
* *48cells 53cells
33cells
0.00
0.05
0.10
0.15
0.20
0.25
SKF 83959RacloprideSCH 23390
+ + ++- -
- - +
---
Dop
amin
e In
duce
dN
et F
RET
46cells
* *48cells 53cells
33cells
80
These results suggested that the desensitization elicited by each agonist in these neurons
occurred through selective occupancy of the D1 receptor.
3.5 Role of GRK2 in Regulating the D1-D2 Receptor Heteromer Mediated Calcium Signal-
To analyze the mechanism by which GRK2 mediated the D1-D2 receptor heteromer calcium
signal desensitization, GRK2 was transiently transfected into the HEK 293T D1-D2 receptor
heteromer stable cell line in increasing concentrations which led to a progressive attenuation of
the calcium signal when activated with dopamine (Fig 3-12A, bars 3-6 and Fig 3-12B). No
significant decrease in this calcium signal was observed when cells were transfected with the
empty vector pcDNA3. The addition of ATP to the GRK2 transfected cells to activate
endogenously expressed purinergic receptors that couple to Gq, also demonstrated a progressive
decline in the ATP mediated calcium signal, indicating the GRK2 was active in these cells (Fig
3-12C, bars 3-6).
3.5.1 Evaluation of GRK2 functional domains in regulating the D1-D2 receptor heteromer
mediated calcium signal- The GRK2 crystal structure confirms that it is composed of three
functional domains: an amino terminal RGS domain, a central protein kinase domain, and a
carboxyl-terminal, Gβγ PH domain (Lodowski et al., 2003). To determine whether the calcium
signal attenuation was due to catalytic or RGS activity, GRK2 mutant constructs were transiently
transfected into the HEK 293T D1-D2 receptor heteromer stable cell line.
81
FIGURE 3-12. Increased expression of GRK 2 led to a concentration dependent decrease of the D1-D2 receptor heteromer activated calcium signal. Data represent the percentage of peak fluorescence of the agonist induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor mediated calcium signal with 10 μM dopamine in HEK 293T cells without or with increasing expression of GRK2 (n=4). (B) Immunoblot demonstrating the increasing expression of GRK2 in D1-D2 receptor expressing cells transfected with GRK2 cDNA. GAPDH immunoreactivity was used as a control for protein loading. (C) Activation of the endogenous purinergic receptor mediated calcium signal with 10 μM ATP in HEK 293T cells without or with increasing expression of GRK2 (n=3-4). A significant difference from control is denoted by * = p < 0.05.
A
B51.
3 2.50 8
_
_80
36
GRK2
GAPDH
μg cDNA
C
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]D
opam
ine
Indu
ced
% P
eak
Fluo
resc
ence
* ** *
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]
ATP
Indu
ced
% P
eak
Fluo
resc
ence
**
*
A
B51.
3 2.50 8
_
_80
36
GRK2
GAPDH
μg cDNA51.3 2.50 8
_
_80
36
GRK2
GAPDH_
_80
36
GRK2
GAPDH
μg cDNA
C
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]D
opam
ine
Indu
ced
% P
eak
Fluo
resc
ence
* ** *
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]D
opam
ine
Indu
ced
% P
eak
Fluo
resc
ence
* ** *
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]
ATP
Indu
ced
% P
eak
Fluo
resc
ence
**
*
contro
l
pcDNA3 1.3 2.5 5 8
0
25
50
75
100
GRK2 Plasmid cDNA [μg]
ATP
Indu
ced
% P
eak
Fluo
resc
ence
**
*
82
Transfection of the catalytically inactive GRK2 (GRK2-K220R), previously shown as being
capable of acting in a dominant negative manner to reverse desensitization of some GPCRs
(Claing et al., 2002; Ferguson, 2001; Krupnick and Benovic, 1998), led to a partial but not
complete restoration of the calcium signal following transfection of 2.5 µg or 5 µg cDNA, in
comparison to wild type GRK2 (Fig 3-13A bars 3,4 and 6,7 and Fig 3-13B lanes 3 and 6). Since
expression of the catalytically inactive GRK2 only led to a partial recovery of the calcium signal,
the involvement of the RGS domain of GRK2 was investigated. Expression of a GRK2 mutant
(GRK2-D110A), that lacks the ability to interact with Gq (Sterne-Marr et al., 2003), also led to a
partial but not complete restoration of the signal following transfection of 2.5 µg or 5 µg cDNA
in comparison to wildtype GRK2 (Fig 3-13A bars 3,5 and 6,8 and Fig 3-13B lanes 4 and 7). Co-
expression of a GRK2 double point mutant (GRK2-R106A/K220R) that lacked both catalytic
and RGS function led to full restoration of the calcium signal following transfection of 1µg
cDNA and partial restoration following transfection of 2.5 µg and 5 µg cDNA (Fig 3-13C, bars
3-5 and Fig 3-13D). Taken together, these results indicated that both the RGS and catalytic
domains of GRK2 played a role in inhibiting D1-D2 receptor heteromer signalling after
activation.
The involvement of GRK2 and its mutant constructs in the desensitization of the
dopamine induced calcium signal after agonist pre-treatment was also investigated. Increased
expression of GRK2 led to a significant increase in desensitization of the dopamine induced
signal after pre-treatment with either SKF 83959 or SKF 83822 for 30 min (Fig 3-14A).
Expression of either GRK2-K220R or GRK2-D110A did not lead to any significant changes in
the level of desensitization after pre-treatment with either agonist (Fig 3-14B).
83
FIGURE 3-13. Effect of catalytic domain mutated or RGS domain mutated GRK2 on the D1-D2 receptor heteromer mediated calcium signal. Data represent the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in HEK 293T cells without or with expression of 2.5 μg or 5 μg cDNA for GRK2 (bars 3 and 6), GRK2-K220R (bars 4 and 7) or GRK2 D110A (bars 4 and 8) (n=3-5). (B) Immunoblot demonstrating the increasing expression of GRK2 and mutated constructs in D1-D2 receptor expressing cells. GAPDH immunoreactivity was used as a control for protein loading. (C) Activation of the D1-D2 receptor heteromer mediated calcium signal with 10 μM dopamine in cells without or with increasing expression of GRK2-R106A/K220R (n=3). (D) Immunoblot demonstrating the increasing expression of GRK2-R106A/K220R. GAPDH immunoreactivity was used as a control for protein loading. A significant difference from control is denoted by * = p < 0.05.
A B
C D
GAPDH_36
_80
0 1 5 μg cDNA2.5
R106A-K220R-GRK2
K220R
GRK2
Mock
D110A
D110A
K220R
GRK2
GAPDH_36
_80
2.5μg 5μg
contro
l
pcD
NA3GRK2
K220R
D110A
GRK2
K220R
D110A
0
25
50
75
100
2.5μg 5μg
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
*
*
* *
Control
pcDNA3 1 2.
5 50
25
50
75
100
R106A/K220R Plasmid cDNA [μg]
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
A B
C D
GAPDH_36
_80
0 1 5 μg cDNA2.5
R106A-K220R-GRK2
GAPDH_36
_80
0 1 5 μg cDNA2.5
GAPDH_36_36
_80_80
0 1 5 μg cDNA2.50 1 5 μg cDNA2.5
R106A-K220R-GRK2
K220R
GRK2
Mock
D110A
D110A
K220R
GRK2
GAPDH_36
_80
2.5μg 5μg
K220R
GRK2
Mock
D110A
D110A
K220R
GRK2K22
0R
GRK2
Mock
D110A
D110A
K220R
GRK2
GAPDH_36_36
_80_80
2.5μg 5μg
contro
l
pcD
NA3GRK2
K220R
D110A
GRK2
K220R
D110A
0
25
50
75
100
2.5μg 5μg
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
*
*
* *
contro
l
pcD
NA3GRK2
K220R
D110A
GRK2
K220R
D110A
0
25
50
75
100
2.5μg 5μg
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
*
*
* *
Control
pcDNA3 1 2.
5 50
25
50
75
100
R106A/K220R Plasmid cDNA [μg]
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
Control
pcDNA3 1 2.
5 50
25
50
75
100
R106A/K220R Plasmid cDNA [μg]
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
* *
84
FIGURE 3-14. Effect of GRK2, catalytic domain mutated or RGS domain mutated GRK2 on the D1-D2 receptor heteromer mediated calcium signal after agonist pre-treatment with dopamine agonists for 30 min. Data represent the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in HEK 293T cells following pre-treatment with 1 μM SKF 83959 or SKF 83822 without or with expression of 1.3 μg cDNA for GRK2 (n=3-4). (B) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in cells following pre-treatment with 1 μM SKF 83959 or SKF 83822 without or with expression of 1.3 μg cDNA for GRK2-K220R or GRK2 D110A (n=3-5). A significant difference from control is denoted by * = p < 0.05.
0
25
50
75
100
SKF 83959 SKF 83822
GRK2 K220RGRK2 D110A
--
--
+- +
- -- -
-++
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
A
B
0
25
50
75
100
SKF 83959 SKF 83822
GRK2 - - -+ +
**
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
0
25
50
75
100
SKF 83959 SKF 83822
GRK2 K220RGRK2 D110A
--
--
+- +
- -- -
-++
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
A
B
0
25
50
75
100
SKF 83959 SKF 83822
GRK2 - - -+ +
**
Dop
amin
e In
duce
d%
Pea
k Fl
uore
scen
ce
85
3.5.2 Knockdown of GRK2 in HEK293T cells and striatal neurons- To further validate the
role of GRK2 in desensitization of the calcium signal, endogenous GRK2 was attenuated with
siRNA in both the D1-D2 receptor heteromer stable cell line as well as in striatal neurons.
Transfection of siRNA to silence GRK2 in the D1-D2 receptor heteromer expressing cells
resulted in significant recovery of the calcium signal after pre-treatment with either SKF 83959
or SKF 83822 for 30 min (Fig 3-15A). Furthermore, the knockdown of endogenous GRK2 led to
a significantly higher dopamine induced calcium signal in the absence of agonist pre-treatment in
two of out of the three experiments performed, indicating the contribution of physiological levels
of endogenous GRK2 in regulating the peak height of the dopamine induced calcium signal (3-
15C). The siRNA mediated reduction in expression of GRK2 levels was confirmed by Western
blotting (Fig 3-15B). Knockdown of endogenous GRK2 in the striatal neurons also resulted in
significant recovery of the dopamine induced calcium signal after exposure to either SKF 83959
or SKF 83822 for 30 min (Fig 3-16A). The siRNA mediated reduction in expression of GRK2
levels in the striatal neurons was confirmed by Western blotting (Fig 3-16B).
3.5.3 D1-D2 receptor heteromer interaction with GRK2- To examine whether the agonists
induced recruitment of GRK2 to the D1-D2 receptor heteromer, we performed
immunocytochemistry on striatal neurons. Pre-treatment of the striatal neurons with 100 nM
dopamine, SKF 83959 or SKF 83822 for 5 min each led to a similar extent of GRK2
relocalization in a punctuate distribution, indicating GRK2 reactivity induced by these agonists
(Fig 3-17).
86
FIGURE 3-15. Decreased expression of GRK2 by siRNA led to significant recovery of the D1-D2 receptor heteromer mediated calcium signal after pre-treatment with either SKF 83959 or SKF 83822 in the HEK 293T D1-D2 receptor heteromer stable cell line. Data represent the percentage of peak fluorescence (A and B) or AFU (C) of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. A.F.U. = absolute fluorescence units. (A) Desensitization of the calcium signal elicited by dopamine following pre-treatment with 1μM SKF 83959 or 1μM SKF 83822 for 30 min after expression of either non silencing (-) or GRK2 siRNA (+) (n=3). (B) Immunoblot demonstrating the decreased expression of GRK2. GAPDH immunoreactivity was used as a control for protein loading. (C) Dopamine induced calcium signal after expression of either non silencing (-) or GRK2 siRNA (+) (n=2).
A
B
0
25
50
75
100SKF 83959 SKF 83822
* *
GRK2 siRNA - -+ +D
opam
ine
Indu
ced
% P
eak
Fluo
resc
ence
__ +
36 _GAPDH
siRNA
80 GRK2
0
25000
50000
75000
100000
GRK2 siRNA - +
Dop
amin
e In
duce
dAF
U
C
A
B
0
25
50
75
100SKF 83959 SKF 83822
* *
GRK2 siRNA - -+ +D
opam
ine
Indu
ced
% P
eak
Fluo
resc
ence
__ +
36 _GAPDH
siRNA
80 GRK2__ +
36 _GAPDH
siRNA
80 GRK2
0
25000
50000
75000
100000
GRK2 siRNA - +
Dop
amin
e In
duce
dAF
U
C
87
FIGURE 3-16. Decreased expression of GRK2 by siRNA led to significant recovery of the D1-D2 receptor heteromer mediated calcium signal after pre-treatment with either SKF 83959 or SKF 83822 in striatal neurons. Data represent the percentage of peak FRET levels of the dopamine induced calcium signal. Peak FRET levels are the means ± S.E.M. from a range of 35 to 60 neurons from a total of 3 experiments. (A) Desensitization of the calcium signal elicited by dopamine following pre-treatment with 100 nM SKF 83959 or 100 nM SKF 83822 for 30 min after expression of either non silencing (-) or GRK2 siRNA (+). (B) Immunoblot demonstrating the decreased expression of GRK2 in striatal neurons. GAPDH immunoreactivity was used as a control for protein loading. A significant difference from control (no-pre-treatment) is denoted by * = p < 0.05.
0
25
50
75
100
GRK2 siRNA
SKF 83959 SKF 83822
- -+ +
* *
Dopa
min
e In
duce
d%
Net
FR
ET
A B
_80
_36 GAPDH
siRNA _ +GRK2
0
25
50
75
100
GRK2 siRNA
SKF 83959 SKF 83822
- -+ +
* *
Dopa
min
e In
duce
d%
Net
FR
ET
A B
_80
_36 GAPDH
siRNA _ +GRK2
_80
_36 GAPDH
siRNA _ +GRK2
_80
_36 GAPDH
siRNA _ +GRK2
88
FIGURE 3-17. Immunocytochemistry of striatal neurons in culture showing endogenously expressed GRK2 localization before (control) and after exposure to either 100 nM dopamine, SKF 83959 or SKF 83822 for 5 min. Arrow indicates distinct punctate.
Control
10μM
SKF 83822
SKF 83959
Dopamine
Control
10μM
Control
10μM10μM
SKF 83822SKF 83822
SKF 83959SKF 83959SKF 83959
DopamineDopamine
89
To determine whether GRK2 physically interacted with the D1-D2 receptor heteromeric
complex, we performed co-immunoprecipitation studies. After 5 min agonist treatment of HEK
293T cells stably expressing both the HA-D1 and FLAG-D2 receptors, the FLAG-D2 receptor
was immunoprecipitated from a P2 membrane preparation. In addition to the D1 receptor at
55kDa, immunoblotting of this preparation revealed a band corresponding to GRK2 at 80kDa
for each agonist treatment that was denser than the control band (no agonist treatment) (Fig 3-
18), suggesting an agonist induced increase in the physical association between the D1-D2
receptor heteromeric complex and GRK2. To measure the extent of GRK2 recruitment, the
GRK2 immunoblot was normalized to the amount of FLAG-D2 receptor immunoprecipitated for
vehicle and each agonist treatment. There was 350%, 270%, and 168% increases in GRK2
precipitation with the FLAG-D2 receptor compared to control for treatments with dopamine,
SKF 83959 and SKF 83822, respectively.
Moreover, to lend further support to the notion of a direct agonist induced interaction
between the D1-D2 receptor heteromer and GRK2, a BRET assay was performed with HEK
293T cells transfected with Rluc-D1 receptor, D2 receptor and GFP-GRK2, or GFP cDNAs.
Treatment with 1 μM dopamine, SKF 83822, or SKF 83959 for 1 min led to a rapid rise in the
BRET ratio between Rluc-D1 receptor and GFP-GRK2 in comparison to control (no agonist
treatment) (Fig 3-19A) and declined within 10 min of agonist exposure (Fig 3-19B). This
indicated a rapid agonist induced association (proximity <100Å) between Rluc-D1 receptor and
GFP-GRK2.
90
FIGURE 3-18. Co-immunoprecipitation of HA-D1 and GRK2 with FLAG-D2 receptor from P2 membranes expressing FLAG-D2 receptor, HA-D1 receptor and GRK2 after the HEK 293T cells were treated with vehicle (control), 1 μM dopamine, SKF 83959, or SKF 83822 for 5 min. IP=Immunoprecipitation with FLAG antibody. IB=Immunoblot with HA or GRK2 antibody.
IB: GRK2 C
ontr
ol
Dop
amin
e
SKF
8395
9
SKF
8382
2
- 80 IB: HA-D1
Con
trol
Dop
amin
e
SKF
8395
9
SKF
8382
2
- 55
IP: FLAG-D2
91
FIGURE 3-19. BRET detection of Rluc-D1 and GFP-GRK2 interaction after either 1 min treatment (A) or 10 min treatment (B) with vehicle (control), 1 μM dopamine, SKF 83959, or SKF 83822. Values shown are the means ± S.E.M. of n=3 experiments. A significant difference from control is denoted by * = p < 0.05.
Control
Dopamine
SKF8395
9
SKF8382
20.00
0.01
0.02
0.03
0.04
0.05
**
*
BR
ET R
atio
Control
Dopamine
SKF8395
9
SKF8382
2-0.01
0.00
0.01
0.02
0.03
0.04
0.05
BR
ET R
atio
A
B
Control
Dopamine
SKF8395
9
SKF8382
20.00
0.01
0.02
0.03
0.04
0.05
**
*
BR
ET R
atio
Control
Dopamine
SKF8395
9
SKF8382
2-0.01
0.00
0.01
0.02
0.03
0.04
0.05
BR
ET R
atio
A
B
92
3.6 Resensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal in
HEK293T Cells- To study resensitization of the calcium signal, the HEK 293T cells co-
expressing D1 and D2 receptors were pre-treated with 1 μM dopamine or SKF 83959 for 30 min,
then was washed off followed by incubation at 37oC for up to 3 hrs before activation of the
calcium signal with dopamine. The desensitized response significantly resensitized within 1.5 hr
and 3 hr of dopamine removal but not SKF 83959 removal (Fig 3-20 A and B). To determine if
protein synthesis played a role in the recovery of the desensitized response, the protein synthesis
inhibitor, cycloheximide, was added to the cells during the 3 hr incubation after dopamine and
SKF 83959 wash off. No significant difference in the level of the calcium signal was observed in
the presence and absence of cycloheximide, indicating protein synthesis was not necessary for
recovery of the signal (Fig 3-20 A and B).
3.7 Internalization and Recycling of D1 and D2 receptors following treatment with
dopamine or SKF 83959- To investigate the intracellular trafficking of D1 and D2 receptors
following agonist treatment, immunocytochemistry was performed with the HEK 293T cell line
stably expressing the D1 and D2 receptors. In the absence of agonist treatment, the D1 receptor
was localized primarily to the cell surface and the D2 receptor was expressed both intracellularly
and at the cell surface (Fig 3-21). To study internalization of the receptors, the cells were treated
with 1 μM dopamine or SKF 83959 for 30 min. Treatment with either agonist induced
significant internalization of both the D1 and D2 receptors (Fig 3-21 and 3-22). To determine
whether the receptors recycled back to the cell surface, the cells were monitored either 1.5 hr or
3 hr after agonist wash off. For dopamine treated cells, the D1 and D2 receptor reappeared at the
cell surface within 1.5 hr and 3 hr of dopamine removal (Fig 3-21), suggesting receptors treated
with dopamine could recycle.
93
FIGURE 3-20. Resensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal. Cells were pre-treated with 1 μM dopamine (A) or SKF 83959 (B) for 30 min and the calcium signal was activated either immediately, 1.5 hr, or 3 hr after agonist wash off in the absence and presence of cycloheximide (35 μM). Values shown are the means ± S.E.M. of n=3-6 experiments. A significant difference from control (no pretreatment) is denoted by * = p < 0.05.
None Dopamine Dopamine Dopamine Dopamine0
25
50
75
100
PretreatmentRecovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hrCycloheximide +
*D
opam
ine
Indu
ced
% P
eak
Fluo
rese
nce
None 83959 83959 83959 83959 0
25
50
75
100
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
PretreatmentRecovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hrCycloheximide +
* * **
A
B
None Dopamine Dopamine Dopamine Dopamine0
25
50
75
100
PretreatmentRecovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hrCycloheximide +
*D
opam
ine
Indu
ced
% P
eak
Fluo
rese
nce
None 83959 83959 83959 83959 0
25
50
75
100
Dop
amin
e In
duce
d%
Pea
k Fl
uore
senc
e
PretreatmentRecovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hrCycloheximide +
* * **
A
B
94
FIGURE 3-21. Trafficking of D1 and D2 receptors after treatment with dopamine in HEK 293T cells stably expressing D1 and D2 receptors. Immunocytochemistry displaying D1 (green), D2 (red), or both receptors (merged) before and after 30 min treatment with 1 μM dopamine as well as 1.5hr and 3hr after dopamine wash off. Images shown are representatives from a range of 93 to 143 cell images.
D1 D2 Merged
Control
Dopamine 30min Treatment
1.5hr Recovery
3hr Recovery
95
FIGURE 3-22.Trafficking of D1 and D2 receptors after treatment with SKF 83959 in HEK 293T cells stably expressing D1 and D2 receptors. Immunocytochemistry displaying D1 (green), D2 (red), or both receptors (merged) before and after 30 min treatment with 1 μM SKF 83959 as well as 1.5hr and 3hr after SKF 83959 wash off. Images shown are representatives from a range of 112 to 167 cell images.
D1 D2 Merged
Control
SKF 83959 30min Treatment
1.5hr Recovery
3hr Recovery
96
In contrast, in cells treated with SKF 83959, the D1 or D2 receptor did not reappear at the cell
surface within 1.5 hr or 3 hr of agonist removal (Fig 3-22), suggesting SKF 83959 treatment did
not result in recycling of these receptors. Taken together, these results suggest that trafficking of
the D1-D2 receptor heteromer is determined by specific ligand exposure.
To quantify D1 and D2 receptor internalization, radioligand binding assays using HEK
293T whole cells were performed with [3H]SCH 23390 or [3H]Raclopride to measure whole cell
surface binding of either the D1 or D2 receptor, respectively. To ensure that [3H]SCH 23390 or
[3H]Raclopride measured cell surface receptors, the assay was conducted at 4ºC and specific
binding to cell surface receptors was calculated by the displacement of radioligand by 100 μM
dopamine. Since dopamine does not cross the cell membrane it would only displace radioligand
from receptors on the cell surface. Non-specific binding accounted for approximately 5% of total
bound ligand. Treatment with SKF 83959 for 5 min or 30 min resulted in significant
internalization of the D1 receptor in cells expressing the D1 receptor alone (68.5 ± 12.8% for 5
min and 75.6 ± 3.3% for 30 min) or cells expressing both the D1 and D2 receptors (67.5 ± 17.5%
for 5 min and 67.7 ± 7.7% for 30 min), indicating maximum D1 receptor internalization occurred
within 5 min of agonist stimulation (Fig 3-23). Treatment with dopamine for 30 min also resulted
internalization of the D1 receptor in cells expressing the D1 receptor alone (26.2 ± 5.0%) or cells
expressing both the D1 and D2 receptors (20.6 ± 2.9%), but it was significantly less than what
was observed with SKF 83959 (Fig 3-23). Treatment with either agonist also resulted in
internalization of the D2 receptor co-expressed with the D1 receptor, but results were
inconsistent and therefore are not reported here.
97
FIGURE 3-23. Agonist induced internalization of the D1 receptor in HEK 293T cells expressing the D1 receptor alone or co-expressing both the D1 and D2 receptors. Cells were treated with 100 nM SKF 83959 for 5 min or 30 min or 1μM dopamine for 30 min. Percentage internalization represents the loss of [3H]SCH 23390 binding from the cell surface of intact cells after agonist treatment compared with vehicle treated controls. Values shown are the means ± S.E.M. of n=3-6 experiments. A significant difference from treatment with SKF 83959 is denoted by * = p < 0.05.
D1 D1-D2 D1 D1-D2 D1 D1-D20
25
50
75
100 SKF 83959
Dopamine
5 min 30 min
30 min*
*
% D
1 R
ecep
tor
Inte
rnal
izat
ion
of C
ontr
ol
98
4 DISCUSSION
4.1 Desensitization of the D1-D2 receptor heteromer mediated calcium signal
The discovery of the D1-D2 receptor heteromer Gq-mediated calcium signal is important
since by this mechanism activation of dopamine receptors is directly linked to calcium signaling.
Given that there is localization of the D1-D2 receptor heteromer throughout the basal ganglia, it
is essential to determine how this complex and its signal are regulated. Termination of a signal
by receptor desensitization is an important component of GPCR signaling. The first study
investigating the desensitization of the D1-D2 receptor complex was reported in 2007 (So et al.,
2007). This report demonstrated that the D1-D2 receptor heteromer mediated calcium signal
rapidly desensitized within 2 minutes by prior exposure to dopamine or selective D1 or D2
agonists in heterologous cells. The desensitization was triggered by agonist occupancy of either
receptor subtype, even though co-occupancy of both receptors was necessary for generation of
the calcium signal. Additionally, desensitization of the signal was suggested to occur before the
complex was recruited into vesicles by the endocytic machinery since preventing internalization
of the complex did not decrease the extent of signal desensitization. Furthermore, the
desensitization occurred specifically through a homologous mechanism and was independent of
intracellular calcium store depletion, suggesting the calcium signal desensitization might occur at
the level of the receptor complex. To study potential mediators of D1-D2 receptor heteromer
calcium signal desensitization, several second messenger kinases were tested, but only GRKs 2
or 3 were shown to play a role (So et al., 2007).
My studies have demonstrated the desensitization of the D1-D2 receptor heteromer
mediated calcium signal by agonists that occupied the D1 receptor binding pocket regardless of
99
whether they activated the D1-D2 receptor heteromer induced calcium signal. This
desensitization was shown to be mediated by at least two functional domains of GRK2.
All three agonists with high affinities for the D1 receptor were compared for their ability
to stimulate intracellular calcium release through the D1-D2 receptor heterooligomer. While SKF
83959 and SKF 81297 generated robust, almost equivalent calcium signals, SKF 83822 was
unable to generate a significant calcium signal in either the D1-D2 receptor heteromer expressing
cells or in striatal neurons. We have previously demonstrated that the D1-D2 receptor heteromer
is coupled to Gq which could be activated by SKF 83959 but not by SKF 83822, as shown by
S35GTPγS incorporation into Gq (Rashid et al., 2007). Desensitization of the D1-D2 receptor
heteromer mediated calcium signal occurred not only by exposure to SKF 83959 and SKF 81297
but also by SKF 83822 to a level comparable with the other agonists. This suggested that D1-D2
receptor heteromer activation was not a prerequisite for its desensitization and that a ligand
occupying the binding pocket of one constituent receptor with high affinity could result in signal
desensitization. A heterologous mechanism involving the adenylyl cyclase pathway was not
responsible for this observed desensitization since inhibition of adenylyl cyclase or PKA did not
significantly change the attenuation of the signal by SKF 83822 in striatal neurons. These results
suggested that receptor occupancy by the agonist and its associated conformational changes may
be sufficient for desensitization of the signal without Gq protein activation. Biochemical and
biophysical data suggest that different ligands can indeed induce and/or stabilize subsets of the
multiple active conformations of a receptor (Swaminath et al., 2005; Vilardaga et al., 2005). It is
possible that occupancy of the receptor by SKF 83822 leads to a stabilization of the receptor into
a conformation where it becomes a target for kinases, arrestins or endocytic machinery without
Gq protein activation. In fact, several ligands that recruit arrestin and/or induce receptor
internalization without stimulating G protein signaling have been identified. Receptors for which
100
this has been shown include the angiotensin II type 1 receptor (Wei et al., 2003), β2-
adrenoreceptor (Shenoy et al., 2006), cholecystokinin receptor (Roettger et al., 1997), V2-
vasopressin receptor (Ren et al., 2005) and type 1 parathyroid hormone receptor (Gesty-Palmer
et al., 2006). However, as opposed to these homooligomeric complexes, agonist induced
structural changes within the D1-D2 receptor heterooligomer likely exhibit an increased level of
complexity as a result of the presence of two distinct dopamine receptor subtypes within a single
heteromeric complex, and hence an increased potential for multiple conformational states.
Alternatively, SKF 83822 exposure may have led to activation of GPCR pathways mediated by
other G proteins other than Gq such as Gα12/13 which could have potentially played a role in
the desensitization of the calcium signal.
Desensitization of the calcium signal occurred independent of calcium storage capacity
and exogenous calcium entry, suggesting that there was no impairment of intracellular calcium
kinetics by agonist activation and that desensitization occurred at the receptor level. However,
the involvement of other calcium homeostatic pathways that may have been affected by D1-D2
receptor heteromer activation could not be ruled out. For example, plasma membrane calcium-
ATPases (PMCAs) are a family of calcium pumps that function to maintain low concentrations
of cytosolic calcium by responding to elevations in calcium following calcium release from
intracellular organelles or after the influx of extracellular calcium (Di Leva et al., 2008). These
PMCAs could potentially have been upregulated by D1-D2 receptor heteromer activation and
therefore played a role in the observed decreased calcium signal after agonist pre-treatment.
To investigate a possible mechanism responsible for D1-D2 receptor heteromer mediated
calcium signal desensitization at the receptor level, GRK2 and its mutant constructs were
utilized. Increased expression of GRK2 led to a concentration dependent decrease of the calcium
signal and knockdown of GRK2 by siRNA led to an increase in the calcium signal. The GRK2
101
constructs that were catalytically inactive or RGS mutated each led to a partial reversal of the
GRK2 calcium signal effect suggesting that both GRK2 domains were involved and thus GRK2
had a dual role in mediating calcium signal desensitization. This is the first demonstration of
GRK2 having a bifunctional role in regulating a GPCR heterooligomer where the constituent
receptor homooligomers are not coupled to the Gq protein.
The lowest concentration of GRK2-R106A/K220R, which lacked both the catalytic and
RGS functions, led to a near complete recovery of the calcium signal, providing further evidence
that both GRK2 domains were involved in the calcium signal regulation. However, expression of
GRK2-R106A/K220R at higher concentrations was not able to completely restore the calcium
signal. Although GRK2-R106A/K220R contains a mutation within its RGS domain resulting in
prevention of Gq binding it is possible that it still retains the ability to bind the D1-D2 receptor
heteromeric complex. Indeed, a recent study has demonstrated that GRK2 and several of its
mutants including the catalytically inactive and RGS mutated GRK2 constructs were able to co-
immunoprecipitate with the D2 receptor homooligomer (Namkung et al., 2009b). A similar
phenomenon has been demonstrated for the metabotropic glutamate receptor 1 (mGluR1) where
GRK2 mutants impaired in Gq binding could still bind avidly to the mGluR1 receptor (Dhami et
al., 2004). Thus, it can be postulated that the failure of higher concentrations of GRK2-
R106A/K220R to completely restore calcium signaling could be attributed to steric hindrance of
receptor-Gq coupling caused by GRK2-R106A/K220R binding to the D1-D2 receptor
heteromeric complex. My results are consistent with what has been reported for the Gq coupled
H1 histamine receptor, where although lower concentrations of the GRK2 double mutant
resulted in little inhibition of agonist induced inositol phosphate production, higher
concentrations showed a partial inhibitory effect (Iwata et al., 2005). Thus, it is possible that a
102
similar process occurred in the present system where excessive binding of GRK2-R106A/K220R
to the D1-D2 receptor heteromer resulted in the attenuation of inherent receptor Gq interactions.
The involvement of GRK2 in calcium signal desensitization after agonist pre-treatment
with either SKF 83959 or SKF 83822 was confirmed by both increasing and decreasing the
expression of GRK2 in D1-D2 receptor heteromer expressing cells as well as by significant
knockdown of endogenous GRK2 in primary culture neurons. Since SKF 83822 does not
activate the calcium signal but has structural similarity to SKF 83959, these results suggest that
GRK2 may be involved in receptor occupancy mediated calcium desensitization by either SKF
83822 or SKF 83959. Accordingly, agonist induced recruitment of GRK2 to the D1-D2 receptor
heteromer was suggested by the redistribution of GRK2 to the cell periphery as well as
significant co-immunoprecipitation of both GRK2 and the D1 receptor with the D2 receptor after
agonist treatment. Although the co-immunoprecipitation data indicated that SKF 83822 resulted
in less GRK2 recruitment than dopamine or SKF 83959, all three agonists still elicited equivalent
levels of desensitization of the D1-D2 receptor heteromer mediated calcium signal. Furthermore,
there was a significant BRET signal between the D1 receptor and GRK2 in the presence of the
D2 receptor, indicating a physical association between the two proteins which was enhanced by
all three agonists.
Expression of either GRK2-K220R or GRK2-D110A did not attenuate the desensitization
of the calcium signal elicited by exposure to either agonist. In contrast, in the absence of agonist
pre-treatment the expression of either GRK2 construct each led to a partial reversal of the GRK2
attenuated calcium signal. It is possible that the presence of agonist resulted in a greater
recruitment of endogenous GRK2 to the D1-D2 receptor heteromeric complex in comparison to
the basal state, resulting in increased signal attenuation and therefore may have masked any
calcium signal recovery effects by the GRK2 mutant constructs. Agonist induced recruitment of
103
GRK2 to the D1-D2 receptor heteromer was suggested by immunocytochemistry, co-
immunoprecipitation and BRET assays.
Alternatively, other GRK2 independent mechanisms may also be involved in the calcium
signal attenuation. For example, agonist exposure may induce the formation of a stable receptor
conformation that prevents restoration of the calcium signal or the receptor complex may have
internalized or have been disrupted after agonist pre-treatment and thus may have resulted in a
decreased dopamine induced calcium signal even in the presence of the GRK2 mutant constructs.
While GRK2 mediated desensitization has been reported for both D1 and D2 receptor
homooligomers, only the catalytic activity of GRK2 has been indicated to be important for the
D1 receptor (Jackson et al., 2002; Kim et al., 2004; Lamey et al., 2002). For D2 receptor
regulation, a recent study has provided evidence to suggest a role for the GRK2 carboxyl-
terminal, Gβγ pleckstrin homology domain in addition to catalytic function (Namkung et al.,
2009b). Given this evidence, it is possible that the GRK2 Gβγ pleckstrin homology domain may
also be involved in D1-D2 receptor heteromer regulation and this will be important to
investigate. Interestingly, the GRK2 RGS domain was not involved in suppressing D2 receptor
homooligomer signaling (Namkung et al., 2009b). Thus, my results indicated that D1-D2
receptor heterooligomer signaling regulation by GRK2 was distinct from D1 and D2 receptor
homooligomer regulation, where this dual function involving both the catalytic and RGS
domains of GRK2 in inhibiting signaling has not been reported. Additionally, adenylyl cyclase
activity regulated by D1 and D2 receptor homooligomers is maximal within 15 to 30 min, as
shown by measuring cAMP using competition radioimmuno assays or column chromatography
(Ryman-Rasmussen et al., 2005; Tong et al., 2001), whereas the D1-D2 receptor heteromer
mediated calcium signal peaks within seconds of activation. Thus, the rapid nature of this
calcium signal may require a quick and robust quenching mechanism that cannot be controlled
104
by the GRK2 catalytic activity alone and therefore regulation by both the catalytic and RGS
domains within GRK2 may favor the prompt termination of the D1-D2 receptor heteromer
mediated signal.
Signaling through the D1-D2 receptor heteromeric complex involves activation of the
Gq/calcium pathway. The ability for D1-D2 receptor heteromer to also signal through the
Gs/cAMP pathway cannot be determined since the D1-D2 receptor heteromer response cannot be
completely separated from the D1 receptor homooligomer response in heterologous cells and
therefore it is impossible to separate out the differences between the D1 receptor homooligomer
Gs/cAMP pathway from a potential D1-D2 receptor heteromer activation of the Gs/cAMP
pathway. However, our lab has previously demonstrated that the agonist SKF 83959, which
selectively activates the D1-D2 receptor heteromer, did not activate Gs/olf in striatal membranes,
but activated Gq as shown by incorporation of S35GTPγS (Rashid et al., 2007).
Another potential mechanism for decreased calcium signaling after agonist exposure
could be that agonist occupancy of the receptors triggers the D1-D2 receptor complex to break
apart, thus disrupting and turning off the signal. Although a majority of studies indicate that cell
surface homooligomers remain intact after agonist activation (Babcock et al., 2003; Dinger et al.,
2003; Lee et al., 2000), agonist-dependent dissociation of GPCR oligomers has been suggested
to occur for certain receptors (Cheng and Miller, 2001; Gines et al., 2000). In fact, we recently
demonstrated that long term dopamine exposure disrupted D1-D2 receptor heterooligomer
formation after activation in heterologous cells (O’Dowd et al., 2011) and therefore short term
agonist treatments used in the present experiments could also potentially result in a similar
disruption of the heterooligomer.
105
In summary, I have demonstrated that desensitization of the D1-D2 receptor heteromer
mediated calcium signal occurs by D1 receptor occupancy with or without signal activation and
that GRK2 plays a role in regulating the signal response with at least two distinct functions. My
results provide evidence for an entirely novel mechanism for dopamine D1-D2 receptor
heteromer desensitization that is distinct from mechanisms that have been reported for D1 or D2
receptor homooligomers.
4.2 Internalization of the D1-D2 receptor heteromer and resensitization of the associated
calcium signal
I have demonstrated that the D1-D2 receptor heteromer internalized by pre-treatment
with either dopamine or SKF 83959, which was accompanied by desensitization of the calcium
signal, but recycling of the receptors and resensitization of the calcium signal only occurred by
exposure to dopamine and not SKF 83959.
Differential agonist exposure to the D1-D2 receptor heteromer resulted in different
regulatory pathways for this heteromeric complex. First, although treatment with either
dopamine or SKF 83959 resulted in internalization of the D1-D2 receptor heteromer, results
from radioligand binding assays indicated that SKF 83959 exposure led to significantly more D1
receptor internalization than dopamine pre-treatment in both HEK 293T cells expressing D1
receptors alone and HEK 293T cells co-expressing D1 and D2 receptors. Second, D1 and D2
receptor recycling to the plasma membrane was observed after dopamine removal but not after
SKF 83959 removal even after a prolonged recovery time. Third, resensitization of the calcium
signal was observed after dopamine wash-off but not after SKF 83959 removal; the protein
synthesis inhibitor, cycloheximide, had no effect on the recovery of the calcium signal,
106
indicating protein synthesis was not necessary for resensitization. Taken together, these results
suggest that trafficking of the D1-D2 receptor heteromer and resensitization of its associated
calcium signal are delineated by particular agonist exposure rather than by a defined property of
the D1-D2 receptor heteromer.
At the basal level without agonist exposure, D1 receptors were predominantly at the
cell surface, whereas D2 receptors were localized both intracellularly and at the cell surface,
which is a particular characteristic of the D2 receptor that has been well documented previously
(Fishburn et al., 1995; Prou et al., 2001; Takeuchi and Fukunaga, 2003). The internalized D1 and
D2 receptors returned back to basal localization after dopamine removal. However, a significant
proportion of the confocal images also demonstrated almost complete recycling of D2 receptors
to the cell surface with no D2 receptors being detected intracellularly. This suggests that
internalized D2 receptors may have formed homooligomers with existing intracellular D2
receptors, resulting in recycling of both the agonist internalized D2 receptors and D2 receptors
preexisting in the cytoplasm.
Following endocytosis, D1 receptor homooligomers are reported to predominantly
recycle back to the plasma membrane (Bartlett et al., 2005; Dumartin et al., 1998; Jackson et al.,
2002; Lamey et al., 2002; Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004; Vickery
and von Zastrow, 1999), but D2 receptor homooligomers are documented to predominantly
degrade after dopamine exposure in cells and in neurons (Bartlett et al., 2005). Therefore, these
results suggest that the dopamine induced D2 receptor trafficking profile is not conserved upon
heterooligomerization with the D1 receptor, since D1-D2 receptor heteromer expression resulted
in D2 receptor recycling. However, it is also important to note that the immunocytochemistry
experiments were not performed in the presence of cycloheximide and therefore the involvement
of D2 receptor synthesis cannot be completely ruled out. Yet, protein synthesis was not
107
responsible for resensitization of the calcium signal, thus suggesting recycling of the receptors
occurred in the absence of protein synthesis. The results also suggest that the D1 receptor
homooligomer recycling response is not conserved upon heteromerization with the D2 receptor
after SKF 83959 exposure, since D1-D2 receptor heteromer expression resulted in a lack of D1
receptor recycling after SKF 83959 pre-treatment. Thus, the D1-D2 receptor heterooligomer
appears to adopt the recycling characteristics of the D1 receptor after dopamine exposure and the
properties of the D2 receptor after SKF 83959 exposure.
Although the results demonstrate that both D1 and D2 receptors internalized in response
to dopamine or SKF 83959 pre-treatment, it is not clear if these receptors remained together or
separated once internalized. Additionally, if the D1-D2 receptor heterooligomer did separate into
its constituent receptors after agonist exposure it is not clear if the receptors reformed into a
heterooligomer once they returned to the cell surface after dopamine exposure. Separation of the
internalized D1 and D2 receptors and then reformation into a heterooligomer at the cell surface
after recycling is a possibility since our lab has recently demonstrated that long term dopamine
exposure (4h) can disrupt D1-D2 receptor heterooligomer formation after activation in
heterologous cells and the receptors can reform into a complex at the cell surface (O'Dowd et al.,
2011). Furthermore, if the D1-D2 receptor heterooligomer did separate once internalized,
reformation at the cell surface would be expected since there was complete resensitization of the
D1-D2 receptor heterooligomer mediated calcium signal after dopamine exposure. However,
agonist treatments were tested up to 30 min in my experiments and therefore may not have
resulted in a similar heterooligomer disruption that was observed after long term agonist
exposure. Moreover, separation and reformation characteristics of this heteromer could depend
on specific ligand exposure and therefore could be different after dopamine vs. SKF 83859
exposure.
108
Differences in receptor recycling back to the plasma membrane and resensitization of the
calcium signal after specific agonist exposure may be due to a differential interaction with
arrestins. Dopamine exposure may allow the D1-D2 receptor heterooligomer to behave with
typical Class A GPCR characteristics. Class A receptors do not internalize with the arrestin
protein since it dissociates from the receptor at or near the plasma membrane, allowing for
dephosphorylation in endosomes and rapid recycling back to the plasma membrane resulting in
resensitization of the signal. In contrast, SKF 83959 exposure may cause the D1-D2 receptor
heteromeric complex to behave with typical Class B GPCR characteristics. These GPCRs form
a stable complex with arrestins, internalize with the arrestin protein bound and have been shown
to either recycle slowly, be retained in the endosomal compartment or traffic to lysosomes for
degradation (Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). While this differential
interaction has been demonstrated for different GPCRs, the ability of different ligands to dictate
a Class A or Class B response for the same receptor has only recently been demonstrated (Lee et
al., 2010), but has never been shown for a receptor heterooligomer. It is believed that the
interaction of arrestin with specific phosphorylated residues in the receptor’s carboxyl-terminal
tail is responsible for determining class A or class B characteristics with specific clusters of
residues absent in class A and present in class B GPCRs (Oakley et al., 2001). Exposure to either
dopamine or SKF 83959 may lead to differences in the extent of phosphorylation of the specific
subset of residues in the carboxyl tail of the D1 and/or D2 receptors resulting in changes in the
strength of the arrestin interaction with the receptors.
In summary, I have demonstrated that recycling of ligand induced internalized D1 and D2
receptors and resensitization the D1-D2 receptor heteromer mediated calcium signal occurs after
exposure to dopamine but not SKF 83959. This is the first demonstration of a ligand dictating the
resensitization response of a desensitized signal mediated through a receptor heteromer.
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4.3 Related Studies
This section describes two publications that are related to the D1-D2 receptor heterooligomer to
which I contributed.
1. So CH, Verma V, O’Dowd BF, George SR (2007). Desensitization of the dopamine D1 and D2 receptor heterooligomer mediated calcium signal by agonist occupancy of either receptor. Mol Pharmacol 72:450-62.
Summary:
This was the first study that demonstrated rapid desensitization of the D1-D2 receptor
heterooligomer mediated calcium signal by pre-treatment with dopamine or selective D1 or D2
receptor agonists. The efficacy, potency and rate of signal desensitization differed between
agonists that selectively occupied the D1 or D2 receptors or both receptors simultaneously.
Several second messenger kinases were tested for a role in this desensitization, but only GRK2
and GRK3 were demonstrated to be important.
2. So CH, Verma V, Alijaniaram M, Cheng R, Rashid AJ, O'Dowd BF, George SR (2009). Calcium signaling by dopamine D5 receptor and D5-D2 receptor heterooligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor heterooligomers. Mol Pharmacol 75: 843-54.
Summary:
Since the D5 receptor has structural and sequence homology with the D1 receptor, this report
investigated if the D5 receptor could also interact with the D2 receptor to mediate a calcium
signal. The results demonstrated that D5 and D2 receptors did indeed form heterooligomers and
co-activation resulted in a calcium signal that was Gq and PLC mediated. However, in contrast
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to what was observed for D1 receptors, which activate robust calcium mobilization only within a
complex with D2 receptors, an extensive Gq mediated calcium signal was triggered by D5
receptors expressed alone and this was attenuated by heterooligomer formation with D2
receptors. Additionally, the D5 receptor homooligomer and D5-D2 receptor heteromer mediated
calcium signals were critically dependent on extracellular calcium stores in addition to
intracellular stores, unlike what is observed for D1-D2 receptor heteromers which only trigger
intracellular calcium release.
My contributions to these publications were performing some of the calcium signaling assays
and aiding in the optimization of experiments and analysis of the experimental results.
4.4 Novel Findings and General Conclusions
Because D1-D2 receptor complexes are linked to calcium signaling and exist in vivo, it is
of major importance then to determine their regulation. Although much is known about the
mechanisms mediating D1 and D2 receptor homooligomers, the regulation of the D1-D2
receptor heteromer was largely unexplored when I began my PhD studies. My work has
identified some of the regulatory properties of the D1-D2 receptor heterooligomer and
demonstrated mechanisms that are distinct from its constituent receptors. A summary of the
novel findings and general conclusions generated by my work is provided below.
1. Desensitization of the D1-D2 receptor heteromer mediated calcium signal occurs by D1
receptor occupancy with or without signal activation.
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By performing calcium mobilization assays in both heterologous cells and cultured striatal
neurons, my studies have demonstrated that agonists occupying the D1 receptor binding pocket
regardless of whether they activated the D1-D2 receptor heteromer mediated calcium signal
resulted in significant desensitization of the signal. These results indicated that D1-D2 receptor
heteromer activation was not a prerequisite for desensitization of its signal and occupancy of the
receptor binding pocket with its associated conformational changes could result in decreased
signaling. This desensitization was mediated through selective occupancy of the D1 receptor
since reduction of the calcium signal was unchanged in the presence of the D2 antagonist,
raclopride, but abolished in the presence of the D1 antagonist, SCH 23390. Moreover,
pretreatment with both a D1 agonist and D2 agonist did not result in a further degree of
desensitization of the calcium signal in comparison to a D1 agonist alone, confirming the
desensitization was elicited through the D1 receptor.
The discrete signaling effects of SKF 83822 could have significant implications for
dopaminergic signaling in vivo, since this agonist could result in activation of D1 receptor
homooligomer signaling while simultaneously attenuating D1-D2 receptor heteromeric signaling
and therefore alter the general tone of dopamine transmission.
Pharmacological modification of dopaminergic signaling is used as a common
therapeutic tool in the treatment of many dopamine related diseases, but there are still currently
many undesirable side effects associated with these agents. Therefore, research still continues to
thrive in order to discover novel therapies with less unwanted side effects. Research development
in agonists that exhibit functional selectivity is a relatively new area of research and refers to
agonists that can selectively modify signal transduction pathways through a single receptor
isoform, depending on the effector pathway coupled to that receptor (Ryman-Rasmussen et al.,
2007). Given that occupancy of the D1 receptor by SKF 83822 within the D1-D2 receptor
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heterooligomer induces GRK2 recruitment, this suggests that arrestins may be involved in the
regulation and these arrestins may also possibly lead to their own signaling pathway, as has been
shown for other GPCR homooligomers (Gesty-Palmer et al., 2006; Ren et al., 2005; Shenoy et
al., 2006). Thus, my results suggest that SKF 83822 could potentially be functionally selective as
an antagonist for the D1-D2 receptor heterooligomer since it attenuates the associated calcium
signal but may lead to a simultaneous arrestin mediated signal as a potential agonist of this
pathway.
Overall, these results add to the complexity of GPCR heteromeric signaling by
demonstrating the potential for unique conformations within a heterooligomer by selective
agonists that can lead to altered signaling pathways, different from the constituent receptor
homooligomers.
2. GRK2 regulates the D1-D2 receptor heteromer by distinct functions.
Calcium mobilization assays in heterologous cells and cultured striatal neurons demonstrated
that GRK2 had a dual role in regulating the D1-D2 receptor heteromer mediated calcium signal.
The contribution of both catalytic and Gq binding functions of GRK2 in regulating D1-D2
receptor heterooligomer signaling not only showed the significance of different multi-function
domains within GRK2, but also demonstrated unique D1-D2 receptor heterooligomer regulation
in comparison to its constituent receptors, findings which support the potential for distinct
regulation of other GPCR heterooligomers by GRK2 as well.
The attenuation of the D1-D2 receptor heteromer mediated calcium signal by GRK2 with
multiple functions adds to the complexity of how this receptor complex is regulated in vivo. In
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addition to providing a quick quenching mechanism, dual functioning by GRK2 could also
enable different levels of attenuation of the calcium signal by potentially only using one
functional domain as opposed to both; regulation by a single function could also lead to changes
in the trafficking and responsiveness of the D1-D2 receptor complex. For example, a reduction in
signaling caused solely by Gq binding functions without catalytic action suggests the endocytic
machinery normally associated with phosphorylation, such as arrestins and clathrin, may not be
involved, thus resulting in changes of the D1-D2 receptor heterooligomer internalization profile.
3. Agonist exposure to the D1-D2 receptor heteromer resulted in recruitment of GRK2 and
direct interaction with GRK2.
Results from Immunocytochemistry, co-immunoprecipitation, and BRET assays suggested that
pretreatment with selective agonists which occupied the D1 or both D1 and D2 receptor binding
pockets regardless of whether they activated the D1-D2 receptor heteromer mediated calcium
signal could lead to an increased GRK2 interaction with the D1-D2 receptor heteromeric
complex. These findings suggest that agonist occupancy of receptors with the D1-D2 receptor
heteroolgiomer is sufficient to induce desensitization through a GRK2 mediated mechanism.
4. The D1-D2 receptor heteromeric complex internalized after dopamine exposure and the
associated calcium signal fully resensitized.
By performing calcium mobilization assays and immunocytochemistry, I have demonstrated that
the D1-D2 receptor heteromer mediated calcium signal not only desensitized after exposure to
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dopamine, but also that the receptor complex internalized followed by receptor recycling back to
the plasma membrane allowing for resensitization of the signal.
Given that the dopamine induced D2 receptor degradative fate is not conserved upon
heteromerization with the D1 receptor, where it appears to recycle rather than degrade, could
have implications with regards to dopaminergic signaling in vivo. Dopamine exposure would
presumably result in differential trafficking of the D2 receptor within the D1-D2 receptor
heteromer vs. the D2 receptor expressed alone leading to changes in dopamine transmission
throughout the brain. The potential long term consequences on dopamine receptor transmission
would depend on the extent of D1-D2 receptor heteromer activation vs. D2 receptor
homooligomer activation within discrete brain regions. For example, repeated dopamine
activation of this receptor complex in the nucleus accumbens, a brain region that demonstrated
the highest percentage of D1-D2 receptor heterooligomer expression in the adult rat (Perreault et
al., 2010), could possibly lead to a shift from D2 receptor homooligomer signaling to D1-D2
receptor heteromeric signaling over time, since the dopamine activated receptor heteromer would
seemingly recycle resulting in a rapidly restored signal, in constrast to D2 receptor
homooligomers that would presumably not recycle and therefore restoration of signaling would
take much longer.
5. The D1-D2 receptor complex internalized after SKF 83959 exposure, but the receptors
did not recycle back to the cell surface.
In contrast to dopamine, it appears that SKF 83959 exposure does not lead to recycling of the
D1-D2 receptor heteromer or resensitization of the signal even after a prolonged recovery time.
These results suggest that exposure to this drug, which selectively activates the D1-D2 receptor
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heteromer, in vivo would lead to changes in the balance of endogenous dopaminergic signaling
since D1 and D2 receptor homooligomers would presumably be unaffected by SKF 83959 but
trafficking and responsiveness of the D1-D2 receptor heteromer would be decreased, thus
leading to long term changes in dopamine transmission. Additionally, the ability for SKF 83959
exposure to possibly lead to decreased D1-D2 receptor heteromer signaling over time, suggests
that it could potentially be used as a pharmacological tool in order to discern how this D1-D2
receptor heteromer mediated signal contributes to neuronal function in vivo.
4.5 Significance and Future Studies
Since there is localization of the D1-D2 receptor heteromer throughout the basal ganglia
(Perreault et al., 2010), furthering the understanding of its activation and regulatory mechanisms
will not only increase our knowledge of how this complex behaves but also may enhance our
understanding of neuropsychiatric diseases in which this receptor heterooligomer may play role.
The functional implications of this unique signaling complex are only beginning to be
understood, but already suggest a role for this D1-D2 receptor heterooligomer in
neuropsychiatric disorders, such as schizophrenia. Schizophrenia has been linked to increased
dopamine transmission and abnormal calcium signaling has been proposed to constitute the
central unifying factor that is responsible for the psychopathology of this disorder (Lidow, 2003).
This D1-D2 receptor heteromer directly links dopamine receptors to calcium signaling providing
a potential mechanism to bridge these two streams of evidence. Additionally, activation of the
D1-D2 receptor heteromer resulted in increased CaMKII activation as well as BDNF expression
(Hasbi et al., 2009; Rashid et al., 2007), two proteins that have been linked to schizophrenia
(Carlino et al., 2011; Jindal et al., 2010; Novak and Seeman, 2010; Weickert et al., 2003; Wong
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et al., 2010). Furthermore, there was an increased proportion of the D1-D2 receptor heteromer in
the agonist detected high affinity state that was observed either in rat striatum after chronic
treatment with amphetamine or in human schizophrenia brain basal ganglia (Perreault et al.,
2010). The D1-D2 receptor heteromer has also been suggested to be involved in the pathology of
depression, since coupling between D1 and D2 receptors was increased in postmortem brain of
subjects suffering from depression and disruption of the D1-D2 receptor complex elicited anti-
depressant like effects in rats (Pei et al., 2010).
The potential role for this D1-D2 receptor complex in disorders such as schizophrenia
suggest it would be of clinical relevance to eventually design drugs that specifically target D1-
D2 receptor heterooligomers in order to aid in the treatment of schizophrenia and/or other
neuropsychiatric disorders. Determining precisely how D1-D2 receptor heteromer intracellular
signaling is regulated will not only increase our understanding of the available repertoire of
dopamine receptor signaling pathways but also enable investigation of how these signaling
pathways may be disrupted in pathogenic states such as schizophrenia. However, further research
is still required to understand the full complement of D1-D2 receptor heteromeric regulation. A
summary of potential future studies that can be performed is provided below.
Future Studies
1. Desensitization mechanisms associated with the D1-D2 receptor heteromer mediated
calcium signal.
My studies have demonstrated that agonists occupying the D1 receptor binding pocket regardless
of whether they activated the D1-D2 receptor heteromer-mediated calcium signal resulted in
significant desensitization of the signal and recruitment of GRK2. However, further studies to
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elucidate mechanisms involved in regulating the D1-D2 receptor heteromeric complex and its
associated calcium signal could be done.
A) Although the coimmunoprecipitation and BRET data suggest a direct agonist induced
interaction between the D1-D2 receptor heteromer and GRK2, other techniques, such as
bimolecular fluorescence complementation, could be performed to demonstrate a more definitive
interaction between GRK2 and both the D1 and D2 receptors. By using bimolecular fluorescence
complementation assays, the D1 and D2 receptor could each be tagged with a complementary
fragment of the donor molecule, R luciferase. Interaction of the D1 and D2 receptor would allow
the two complementary fragments to be brought together allowing for a functional R luciferase
donor molecule that could transfer light energy to an acceptor fluorophore tagged-GRK2 protein
if it was interacting with the D1-D2 receptor heteromeric complex. Furthermore, an additional
control experiment to complement the current coimmunoprecipitation data could be to
immunoprecipitate the D2 receptor from HEK 293T cells only expressing the D2 receptor and
then blot for the GRK2 protein after SKF 83959 treatment.The absence of a band for the GRK2
protein from these D2 receptor expressing cells would help prove that GRK2 is indeed
interacting with D1-D2 receptor heterooligomers, rather than D2 receptor homooligomers, in the
HEK 293T cells stably expressing D1 and D2 receptors.
B) The GRK2 catalytic domain is responsible for its phosphorylation action on receptors. Given
the partial involvement of this domain in regulating the D1-D2 receptor heteromer mediated
calcium signal suggests that GRK2 phosphorylation of the D1-D2 receptor complex may be
involved in the regulatory process. If GRK2 phosphorylates this receptor complex, the
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phosphorylation sites on the D1 and D2 receptors expressed alone may not be conserved upon
D1-D2 receptor heteromer expression or only one receptor within the complex may get
phosphorylated. D1-D2 receptor heteromer phosphorylation by GRK2 could be investigated by
performing phosphorylation assays using HEK 293T cells stably expressing D1 and D2 receptors
and testing for radiolabelled phosphate incorporation on D1 and/or D2 receptors. Additionally,
receptor mutagenesis studies could be performed to elucidate the potential GRK2
phosphorylation sites on the D1 and/or D2 receptors.
C) My studies have demonstrated that both the catalytic and RGS domain of GRK2 are involved
in regulating the D1-D2 receptor heterooligomer. The Gβγ domain of GRK2 has also been
reported to be involved in D2 receptor homooligomer regulation (Namkung et al., 2009b). Thus,
the GRK2 Gβγ PH domain may also be involved in D1-D2 receptor heteromer regulation. To
investigate if the PH domain of GRK2 is involved, the calcium signal could be tested after
expressing either a GRK2 mutant lacking Gβγ binding ability or just the c-terminal PH domain
of GRK2 in cells.
D) Although I focused my studies on GRK2, GRK3 is also widely distributed throughout the
brain, but at a much lower expression level relative to GRK2 in most brain regions (Arriza et al.,
1992; Erdtmann-Vourliotis et al., 2001). Despite this lower expression level, it could be involved
in regulating the D1-D2 receptor heteromer complex since it was documented to play a role in
the regulation of the D1-D2 receptor complex in heterologous cells (So et al., 2007). This role
for GRK3 in regulating the D1-D2 receptor heteromer could be further explored to investigate if
it regulates the complex at a physiological level and if so, the significance of its domains could
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be determined. Similar to GRK2, the RGS domain of GRK3 has been shown to bind to Gq
proteins and sequester them, therefore GRK3 could also potentially regulate the D1-D2 receptor
complex with multiple domains. The importance of GRK3 could be tested using similar
strategies that demonstrated the involvement of GRK2, including testing of the calcium signal
after expression either wildtype GRK3, GRK3 constructs with mutations in distinct functional
domains, or siRNA to knockdown endogenous GRK3.
E) Since I have demonstrated that the RGS domain of GRK2 is involved in the desensitization of
the calcium signal, it is possible that RGS proteins may also mediate this desensitization. RGS
proteins regulate G protein signaling by limiting the signals generated by GPCRs. They
dramatically increase the rate at which Gα subunits hydrolyze GTP to GDP, a property that
defines them as GTPase activating proteins (Bansal et al., 2007). It has also been shown that
some RGS proteins can diminish Gα mediated signaling by functionally inhibiting Gα effector
coupling, a phenomenon known as “effector antagonism”, where the RGS proteins compete with
effector molecules for binding to Gα subunits making them unavailable for further signaling
(Hepler et al., 1997; Heximer et al., 1997). For example, RGS2 and RGS3 have been shown to
inhibit Gq mediated signaling by effector antagonism in the absence of GTPase acceleration
(Anger et al., 2004). Therefore, it is hypothesized that RGS proteins such as RGS2 or RGS3 may
function to decrease the D1-D2 receptor heteromer mediated calcium signal by binding to Gq
proteins and sequestering them so that they can no longer participate in signaling. In order to test
this hypothesis, the calcium signal could be tested after either over expression of RGS proteins or
reduced expression by using siRNA to knock down endogenous levels.
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2. Internalization mechanisms associated with the D1-D2 receptor heteromer complex.
By performing radioligand binding assays and using confocal imaging, I have demonstrated that
the D1-D2 receptor complex internalizes after exposure to either dopamine or SKF 83959.
However, the mechanisms involved in this internalization are still unexplored.
A) Although I have demonstrated a role for GRK2 in D1-D2 receptor heteromer mediated
calcium signaling desensitization, the involvement of the endocytic machinery, such as arrestins,
could also be investigated. In addition to a role for arrestins in the desensitization and
internalization of the D1-D2 receptor complex after exposure to either SKF 83959 or SKF
83822, arrestin recruitment could potentially lead to its own signaling pathway, such as the
MAPK signaling cascade, even in the absence of G protein activation, as has been shown for
some other GPCR homoligomers (Gesty-Palmer et al., 2006; Ren et al., 2005; Shenoy et al.,
2006; Wei et al., 2003).
It has been demonstrated that the machinery associated with internalization of GPCR
heterooligomers can vary compared to when the receptor is expressed alone (AbdAlla et al.,
2000; Terrillon et al., 2004). Therefore, it is possible that the dynamin-dependent pathway
associated with the internalization of D1 receptor homooligomers may not be conserved upon
heterooligomer formation with the D2 receptor, since D2 receptor internalization, when observed
in certain cell lines, is mediated by dynamin-independent pathways (Vickery and von Zastrow,
1999). Additionally, different subtypes of both dynamin and arrestin proteins exist and therefore
could potentially differ for the D1-D2 receptor heterooligomer in comparison to its constituent
homooligomers.
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A role for proteins such as arrestins and dynamin could be examined by either expressing
wildtype forms, functionally inert mutated forms to knockout wildtype functions dominant
negatively, different subtypes, or siRNA to knockdown endogenous levels of these proteins and
then testing the effects on internalization of the D1-D2 receptor heterooligomer.
B) The D1-D2 receptor heteromer may also internalize through a caveolin-mediated process
since this pathway was shown to be utilized by the D1 receptor expressed alone (Kong et al.,
2007; Kong et al., 2011). The D1 receptor localized to low density caveolin-enriched membrane
domains and associated with caveolin-1 in rat brain. This caveolar based mechanism for
internalization of the D1 receptor may or may not be conserved upon D1-D2 receptor
heterooligomer expression or may also modulate D2 receptor internalization. To determine if the
D1-D2 receptor complex can internalize through this caveolar pathway, caveolin-enriched
fractions could be purified from HEK293T cells co-expressing D1 and D2 receptors by
performing sucrose density gradient centrifugation and then analyzing for D1 and D2 receptor
distribution within these fractions. To test for an interaction between the D1-D2 receptor
heterooligomer and caveolin-1, co-immunoprecipitation and BRET assays could be done with
the heterologous cells co-expressing D1 and D2 receptors. Moreover, rat striatal neurons in
culture could be treated with SKF 83959 to selectively activate the D1-D2 receptor heteromeric
complex at a physiological level, followed by immunocytochemistry to test for internalization of
the receptor complex and co-localization between the receptors and caveolin-1.
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3. D1-D2 receptor heteromer trafficking after agonist induced internalization and
resensitization of the calcium signal.
My studies have demonstrated that specific ligand exposure determined the resensitization
response of the D1-D2 receptor heteromer mediated calcium signal as well as D1 and D2
receptor recycling, but further studies are still required to fully elucidate the mechanisms for
these differential responses.
A) I have demonstrated that exposure to dopamine resulted in recycling of internalized D1 and
D2 receptors and resensitization of the D1-D2 receptor heteromer mediated calcium signal
whereas exposure to SKF 83959 did not result in recycling of internalized D1 and D2 receptors
or resensitization of the calcium signal. This difference could be mediated by a differential
interaction with arrestins where the D1-D2 receptor heterooligomer either dissociates from
arrestin or forms a stable complex with it after ligand exposure. This differential interaction with
arrestins is a general phenomenon that has been shown for GPCR homooligomers designating
them as either Class A, where the receptors internalize without arrestin bound and recycle or
Class B, where the receptors internalize with the arrestin protein bound and do not recycle
(Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). Thus, dopamine pre-treatment
may result in internalization of the D1-D2 receptor complex without the arrestin protein leading
to recycling of the receptors back to the plasma membrane. In contrast, SKF 83959 pre-treatment
may result in internalization of the D1-D2 receptor complex with the arrestin protein still bound
and therefore the receptors are either retained in endosomal compartments or targeted for
degradation.
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To test the interaction with arrestin, its translocation with D1 and D2 receptors after pre-
treatment with either dopamine or SKF 83959 could be monitored by performing
immunocytofluorescence studies in which the fate of the arrestin protein after agonist wash off
and recovery periods could be observed. Additionally, confocal microscopy FRET analysis could
be performed to detect an interaction between the D1-D2 receptor heterooligomer and arrestin
after treatment with either dopamine or SKF83959. The FRET signal could be monitored
immediately after agonist removal as well as after different recovery time points.
B) Since I have demonstrated that D1 and D2 receptor recycling is different after exposure to
either dopamine or SKF 83959, it would be of interest to investigate the trafficking of the
receptors after agonist induced internalization, especially after SKF 83959 exposure since the
receptors did not recycle back to the plasma membrane after a prolonged recovery time. One
group of proteins, known as Rab proteins, are a ras super family of GTPases that control a
variety of important cellular processes, such as endocytosis, trafficking, endosome fusion and
exocytosis (Seachrist and Ferguson, 2003). The Rab proteins have individual isoforms that
localize to the surfaces of distinct membrane bound organelles and therefore can be used as
markers for different endosomes. For example, the Rab 4 protein is mainly localized in early
endosomes and Rab 11 is mainly localized in perinuclear recycling endosomes and the trans-
Golgi network. It is believed that two distinct intracellular systems regulate the recycling of
internalized GPCRs, where one is the Rab 4 mediated rapid recycling pathway and the other is
the Rab 11 mediated slow recycling pathway or trafficking to the trans-Golgi network (Pereira-
Leal and Seabra, 2001; Pfeffer, 2003; Zerial and McBride, 2001).
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Dopamine or SKF 83959 pretreatment could potentially target D1 and D2 receptors to
different endosomes resulting in different recycling pathways. To investigate this possibility, co-
localization of D1 and D2 receptors with different rab proteins, such as Rab 4 and Rab 11, after
either dopamine or SKF 83959 pretreatment could be visualized through fluorescence
microscopy in HEK 293T cells stably expressing both the D1 and D2 receptors. Additionally,
resensitization of the calcium signal could be evaluated after expressing constitutively active or
dominant negative mutants of the Rab proteins.
4.6 Concluding Remarks
My studies demonstrated that the D1-D2 receptor heterooligomer is regulated with unique
characteristics from its constituent receptors and displays regulatory and trafficking properties
that depend on specific ligand exposure. Dopamine receptors play a critical role in physiological
functions through the transmission of extra-cellular signals to the intracellular compartments.
Given that typically, multiple intracellular signaling pathways are activated at a given time,
efficient orchestrating mechanisms are required for appropriate biological outcomes and
therefore defining these mechanisms for this novel D1-D2 receptor heteromer is important for
advancing our knowledge of dopamine receptor signaling and regulation in brain.
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