Basic concepts in G-protein-coupledreceptor homo- andheterodimerization
www.bq.ub.es/recep/franco.htmlwww.rafaelfranco.cat
RAFAEL [email protected]
NIDA minisymposium. SfNSan Diego. November 2007
STOCKHOLMK. FuxeC. IbáñezD. Marcellino
NIH/NIDA N. VolkowS. GoldbergS. FerréA. Woods
MODENAL. AgnatiS. TanganelliP. G. de BenedettiF. Fanelli
BERLINM. Bader
MONTREALM. Bouvier
WAKE-FOREST U.D. Roberts
COIMBRAR. Cunha
ALBACETER. Luján
GLASGOWG. MilliganM. Canals
Centro Investigación Médica Aplicada
Pamplona
Digna Biotech
GPCR classes
NOMENCLATURE
Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic- Metabotropic (G-protein-coupled, GPCR)
Receptor homomerReceptor heteromer
Heteromeric receptor
Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446
Heteromeric receptor: Ach receptor
NOMENCLATURE
Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic- Metabotropic (G-protein-coupled, GPCR)
Receptor homomerReceptor heteromer
Heteromeric receptor
Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446
Receptor homomers
Adenosine A2A
Dopamine D2Dopamine D2 Adenosine A2A Adenosine A2A
Receptor heteromers
Adenosine A2ADopamine D2 NMDADopamine D1
Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446
HETEROMERS AS SENSORS
•1983: Evidence and formulation of the hypothesis: Agnati et al., Neurosc. Letters •1994: DR homomers: Ng et al., Eur. J. Pharmacol. (infected cells)•1995: A1R homomers: Ciruela et al., J. Neurosc. Res. (brain extracts)•Late nineties, early XXI Century: heteromers (opioid, GABA, Dopa/Ado)
Crude IP Avidina
0
20
40
60
80
100
RD
S (%
oft
otal
A2A
R)
Crudeextract
Cellsurface
**
*
**
*
100
50
MW(kDa)
150
100
50
MW(kDa)
150
The A2A
R dimer is the functional specie
Canals et al (2004) J Neurochem 88, 726-734
The C terminal tail of A2A
R is not required for dimerization
A2A R A2A R
Crude
Biotina
IP A2A R
HETERODIMERS: DR/AR
D2R D1R
D2
R-A2A
R receptor heteromers
D1
R-A1
R receptor heteromers
Hillion et al (2002) J Biol Chem
Gines et al (2000) PNAS
Rluc
A B
YFP
Coelenterazine H
470 nm
Rluc
AB
YFP
Coelenterazine H
530 nm
GFP2
A B
YFP
396 nm
510 nm
GFP2
AB
YFP 530 nm
396 nm
BRET FRET
The A2AR/D2 homodimers: BRET: Bioluminiscence Resonance Energy Transfer
0 10 20 30 400.0
0.05
0.1
YFP/Rluc
BR
ET in
crea
se A2A RRluc-D2 RYFP
A2A RRluc-GABABRYFP
BRET SATURATION CURVE
Canals et al (2003) J Biol Chem 278: 46741-49
LOOKING FOR THEBIOCHEMICAL/PHARMACOLOGICAL
“DIMER FINGERPRINT”
IUBCP: Recognition and nomenclature
Alternative nomenclature in:Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446
1 A Coimmunolocalization1 B Coimmunoprecipitation/transgenics2 Specific functional property (Pharmacology/Signalling)3 Knockouts: Loss of function!!!??
IUBCP: Recognition 2 out of 3:
a b
Intramolecular cross-talk in the homomer:Cooperativity
2-R1-R 1-R
2-R1-R 3-R1-R
a b
dc
Intramolecular cross-talk in the heteromer:Cooperativity and/or ?
Looking for the “biochemical fingerprint”of a receptor heteromer
Fingerprint lost in knockout animals
Transfection Radioligand Displacer KD
A1R [3H]R-PIA
R-PIA
Caffeine
1.9±0.4 nM
90±20 M
A2AR [3H]CGS21680 CGS21680
Caffeine
110±30 nM
7±1 M
A1R+A2AR [3H]R-PIA
[3H]CGS21680
R-PIA
Caffeine
CGS21680
Caffeine
1.9±0.3 nM
90±20 M
120±40 nM
90±20 M*
A2AR+D2R [3H]CGS21680 CGS21680
Caffeine
110±40 nM
5±2 M
Different affinities for ligands: Caffeine as adenosine antagonist
A+A+(RR)
A+A(RR)
A(RR)A
A+A+(RR)*
A+A(RR)*
A(RR)*A
L
α L
θα L
α K
μθ K
K
μK
Franco et al (2005) Trends Biochem Sci 30: 360-66
RECEPTOR DIMER MODEL
A + R + G AR+G
A + RG ARG
TERNARY MODEL
A+A+(R–R) A+A(R–R) A(R–R)A
A+A+(R~R) A+A(R~R) A(R~R)A
L α L θα L
K μK
α K μθ K
[ ][ ]
[ ] [ ][ ] [ ]22
22
A)1(A)1(1A)1(2A)1(
RA
⋅+⋅+⋅+⋅++
⋅+⋅+⋅+⋅=
LKLKLLKLK
T
Bound
αθμααθμα
KD1 KD2A2A
RR
RR*
RR
RR*
RR
RR*
Abound = (KD2 A + 2 A2) RT /(KD1 KD2 + KD2 A + A2)
a
b
Dimer Cooperativity Index, Dc:It depends on KD1 and KD2
Casadó et al., Pharmacol Ther In the Press (Dec 2007)
D50 in Drug discovery:A more meaningful
parameter to measure binding potency
compared to IC50 .
Allosterism:Useful parameter to
“measure allosterism”
LOOKING FOR THEFUNCTIONAL
“DIMER FINGERPRINT”
Franco et al Trends Biochemical Sci 28 (2003) 238-243
Fig 4 Clustering adaptor/scaffolding proteins
Signalling A:
Signalling B:
Agonists for the two receptors
Blue agonist “sensitizes” yellow and pink receptors
Blue agonist “desensitizes” yellow and pink receptors
Blue agonist “sensitizes” pink and “desensitizes” yellow
SIGNALLING SIGNALLING
SIGNALLING
SIGNALLINGSIGNALLING
SIGNALLING
Signalling Signalling
Signalling
Fig 3
Franco et al Trends Biochemical Sci 28 (2003) 238-243
0
50
100
150
200
250
300
350
400
450
R-PIA SKF38393 R-PIA+SKF
Naive30 min60 min120 min
cAMP producion via D1R after pretreatment with agonists of A1R and/or D1R
(A1/D1 cotransfected mouse Ltk- fibroblasts)
The intracytoplasmic receptor-receptor cross-talk: ANTAGONISM (A1R/D1R)
Ginés et al (2000) PNAS 97, 8606-11
A Cyclase
GsGi
D2-R
A Cyclase
Gs
D1-R
Ca2+ IP3
D2-RD1-R
Gq
Ca2+ calmodulin kinase IIα(.: increase in accumbens)
Synaptic Plasticity Diacylglycerol
PKC
Rashid et al. (2007) Proc Natl Acad Sci 104, 654-9
Shift to Gq coupling in the D1-D2 receptor heteromer
1.-D1 receptor agonist affinity is enhanced by D3 agonists. This indicates the existence of a synergistic intramembrane receptor-receptor interaction.
2.-Experiments in reserpinized mice showed that D3 receptor stimulation potentiates D1receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. Marcellino et al. (2007) Submitted
D3-RD1-R
D1-D3 Receptor heteromerization
-FRET-BRET-Fingerprint (Intramembrane cross-talk)
Dual regulatory role of Adenosine on Glu release via A1 R/A2A R
Ciruela et al (2006) J Neurosc 26:2080-87
CONCLUSIONS
1. Heteromers exist in natural tissues2. Heteromers are sensors for neurotransmitters 3. Heteromerization modifies:
- biochemical,- pharmacological,- and functional properties of receptors
FUNCTIONAL MODULES IN THE PLASMA MEMBRANE
ADA
mGluR1α
ADA
hsc73
A1
R D1
R
ßArrestincaveolin
A2A
R mGluR5D2
R
α-actinin
homerCa-calmodulin
filamin
ßArrestincaveolin
Class A Rhodopsin likeAminePeptideHormone protein(Rhodopsin
Rhodopsin VertebrateRhodopsin Vertebrate
type 1Rhodopsin Vertebrate
type 2Rhodopsin Vertebrate
type 3Rhodopsin Vertebrate
type 4Rhodopsin Vertebrate
type 5Rhodopsin ArthropodRhodopsin MolluscRhodopsin Other
OlfactoryProstanoidNucleotide-likeCannabisPlatelet activating factorGonadotropin-releasing hormoneThyrotropin-releasing hormone &
SecretagogueMelatoninViralLysosphingolipid & LPA (EDG)Leukotriene B4 receptorClass A Orphan/other
Class B Secretin likeClass C Metabotropic glutamate / pheromoneClass D Fungal pheromoneClass E cAMP receptors (Dictyostelium)Frizzled/Smoothened family