homologous regulation of the heptahelical d1a receptor responsiveness: specific cytoplasmic tail...

Homologous regulation of the heptahelical D1A receptor

responsiveness: specific cytoplasmic tail regions mediate

dopamine-induced phosphorylation, desensitization and

endocytosis

Adele Jackson,1 Rafal M. Iwasiow,1 Ziad Y. Chaar,1 Marie-France Nantel and Mario Tiberi

Ottawa Health Research Institute, and Departments of Medicine/Cellular and Molecular Medicine, University of Ottawa, Ottawa,

Ontario, Canada

Abstract

In the present study, we investigate the role of specific cyto-

plasmic tail (CT) regions of the D1A receptor in mediating

dopamine (DA)-induced phosphorylation, desensitization and

endocytosis. Results obtained in human embryonic kidney

(HEK) cells expressing the wild-type (WT) or truncation forms

(D425, D379 and D351) of the D1A receptor show that

sequences located downstream of Gly379 regulate DA-medi-

ated phosphorylation-dependent desensitization of D1A

receptors. However, the longer truncation mutant D351 failed

to undergo detectable DA-induced phosphorylation while

exhibiting DA-induced desensitization features similar to the

shorter truncation mutant D379. These data potentially suggest

a novel role for a receptor phosphorylation-independent pro-

cess in the DA-promoted D1A subtype desensitization. Our

immunofluorescence data also suggest that sequences

located between Cys351 and Gly379 play an important role in

DA-mediated receptor endocytosis. Additionally, time-course

studies were done in intact cells expressing WT or truncation

receptors to measure the observed rate constant for adenylyl

cyclase (AC) activation or kobs, a parameter linked to the

receptor-G protein coupling status. In agreement with the

desensitization data, D425- and D379-expressing cells exhibit

an increase of kobs in comparison with WT-expressing cells.

Nevertheless, D351-expressing cells, which harbor similar

desensitization features of D379-expressing cells, display no

change in kobs when compared with WT-expressing cells. Our

results suggest that a defective DA-induced endocytosis may

hamper D351 resensitization and concomitant increase in kobs.

Thus, our study showing that specific D1A receptor CT

sequences regulate DA-induced phosphorylation, desensiti-

zation, and endocytosis highlights the underlying molecular

complexity of signaling at dopaminergic synapses.

Keywords: D1A receptor, dopamine, endocytosis, human

embryonic kidney cells, mutagenesis, phosphorylation.

J. Neurochem. (2002) 82, 683–697.

Dopamine (DA) operates in the central nervous system (CNS)

and peripheral tissues through the binding to and activation of

heptahelical receptors that belong to the large family of G

protein-coupled receptors (GPCRs). The heptahelical dop-

aminergic receptors mediate a myriad of physiological effects

in the CNS and peripheral tissues (Missale et al. 1998; Sibley

Received January 22, 2002; revised manuscript received April 26, 2002;

accepted April 29, 2002.

Address correspondence and reprint requests to Mario Tiberi, Ottawa

Health Research Institute, Moses and Rose Loeb Research Centre, 725

Parkdale Avenue, Ottawa, ON, K1Y 4K9, Canada.

E-mail: [email protected] equally to the work.

Abbreviations used: AC, adenylyl cyclase; BSA, bovine serum al-

bumin; CA, [3H]cyclic AMP formed; CA/TU, CA/TU, [3H]cAMP

formed divided by the total uptake CT, cytoplasmic tail; CXCR4,

chemokine CXC receptor 4; D351, a truncated form of the rat D1Areceptor ending at cysteine 351; D379, a truncated form of the rat D1A

receptor ending at glycine 379; D425, a truncated form of the rat D1Areceptor ending at aspartate 425; DA, dopamine; EC50, half-maximal

effective concentration; FBS, fetal bovine serum; GPCR, G protein-

coupled receptor; GRK, G protein-coupled receptor kinase; HA, hem-

agglutinin protein epitope; HEK, human embryonic kidney cells; HRP,

horseradish peroxidase; IBMX, 1-methyl-3-isobutylxanthine; IL3, third

intracellular loop; Kd, equilibrium dissociation constant; kobs, observed

rate constant; MEM, minimum essential medium; NaF, sodium fluor-

ide; PBS, phosphate-buffered saline; PKA, protein kinase A; PMSF,

phenylmethylsulfonyl fluoride; R, receptor number; RIPA, radio-

immunoprecipitation assay; SDS, sodium dodecyl sulfate; TU, total

uptake; WT, wild type.

Journal of Neurochemistry, 2002, 82, 683–697

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 683–697 683

1999). These receptors are grouped into D1-like (D1A/D1,

and D1B/D5) and D2-like (D2short, D2long, D3 and D4)

subtypes, which couple to the stimulation and inhibition of

adenylyl cyclase activity (AC), respectively (Missale et al.

1998). Impairment in dopaminergic activity is the hallmark of

several pathophysiological conditions (Verhoeff 1999; Berke

and Hyman 2000; Carey 2001). A genetic linkage or

association of the various DA receptors with specific

pathophysiological conditions has yet to be clearly demon-

strated (Wong et al. 2000). Meanwhile, data from several

laboratories strongly suggest that alterations in post-transla-

tional modifications of the heptahelical D1A receptor poten-

tially contribute to the etiology of several CNS and peripheral

disorders (Roseboom and Gnegy 1989; Okubo et al. 1997;

Sanada et al. 1999; Dumartin et al. 2000; Zhen et al. 2001).

These observations are substantiated by recent studies

showing that agonist-promoted phosphorylation, desensitiza-

tion and endocytosis of GPCRs are important cellular

processes in regulating specific receptor-mediated physiolo-

gical responses in vivo (Koch et al. 2000; Wess 2000; Nestler

and Landsman 2001; Pierce and Lefkowitz 2001).

Rapid agonist-induced regulation of the D1A receptor

responsiveness has been shown in cellular systems expres-

sing endogenously or ectopically this dopaminergic GPCR

(Balmforth et al. 1990; Chneiweiss et al. 1990; Bates et al.

1991; Zhou et al. 1991; Ofori et al. 1993; Black et al. 1994;

Ng et al. 1994; Ng et al. 1995; Tiberi et al. 1996; Jiang and

Sibley 1999; Vickery and von Zastrow 1999; Gardner et al.

2001). Phosphorylation of heptahelical D1A receptors by

protein kinase A (PKA) and G protein-coupled receptor

kinases (GRKs) has been clearly demonstrated (Zamanillo

et al. 1995; Tiberi et al. 1996; Lewis et al. 1998; Gardner

et al. 2001). Mutagenesis studies suggest that residues within

the third intracellular loop (IL3) and cytoplasmic tail (CT)

regulate agonist-induced D1A receptor desensitization (Jen-

sen et al. 1995; Jiang and Sibley 1999). However, the

specific residues found within these structural determinants

controlling the DA-induced phosphorylation-dependent

desensitization of heptahelical D1A receptors have yet to

be clearly identified. Likewise, the role phosphorylation

plays in agonist-induced D1A receptor endocytosis is

unclear. Indeed, mutation of a PKA site (Thr268) located

on the IL3 of monkey D1A receptor has been shown to

reduce significantly agonist-induced receptor phosphoryla-

tion without any alterations in rapid DA-induced desensiti-

zation and endocytosis (Mason et al. 2002). However,

trafficking to the perinuclear region of cells was inhibited

suggesting a role for Thr268-dependent phosphorylation in

regulating the late sorting of the D1A subtype (Mason et al.

2002). Recent studies have shown that CT sequences govern

the D1A subtype-specific ligand binding and G protein

coupling properties (Iwasiow et al. 1999; Demchyshyn et al.

2000; Jackson et al. 2000; Chaar et al. 2001). Meanwhile,

the functional relationship that exists between the CT of the

D1A receptor and short-term DA-induced phosphorylation,

desensitization, and endocytosis needs to be clarified (Lamey

et al. 2002; Mason et al. 2002). Intriguingly, the goldfish

D1A receptor ortholog displays similar long-term desensiti-

zation features in comparison with its cognate human

equivalent while harboring a significantly shorter CT region

(�80 amino acids) than its mammalian counterparts (Frailet al. 1993). These findings raise an important question with

regard to the role of the CT in mediating agonist-induced

D1A receptor phosphorylation and endocytosis.

In the present study, we aim at investigating the functional

role of the CT of mammalian D1A dopaminergic receptors in

DA-induced phosphorylation, desensitization and endocyto-

sis. We demonstrate that specific CT regions regulate agonist-

induced phosphorylation, desensitization and endocytosis of

the heptahelical D1A receptor. Furthermore, our studies

unravel a potential role for GPCR phosphorylation-independ-

ent desensitization in regulating the D1A subtype respon-

siveness. Finally, our data also suggest that D1A receptor

endocytosis and resensitization potentially controls the

DA-mediatedrateandextentofACactivationinneurons.

Materials and methods

Materials

[32P]Orthophosphate (10 mCi/mL; carrier-free), [3H]adenine (24–

27 Ci/mmol), [14C]cAMP (250–275 mCi/mmol), N-[methyl-3H]

SCH23390 (72–89 Ci/mmol), mouse horseradish peroxidase

(HRP)-linked IgG F(ab¢)2 fragment (from sheep), ECL reagents andBCS liquid scintillation cocktail were purchased from Amersham

Pharmacia Biotech (Baie d’Urfe, Quebec, Canada). DA, flupentixol,

leupeptine, benzamidine, soybean trypsin inhibitor, aprotinin, pepst-

atin A, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride

(NaF), 1-methyl-3-isobutylxanthine (IBMX), cAMP, disodium pyro-

phosphate, 2-mercaptoethanol, sodium dodecyl sulfate (SDS) were

from Sigma/RBI (Oakville, Ontario, Canada). Saponin was obtained

from Acros Organics (Morris Plain, NJ, USA). Rat monoclonal anti-

HA affinity matrix and mouse monoclonal anti-HA antibodies were

purchased from Roche Molecular Biochemicals (Laval, Quebec,

Canada). Human embryonic kidney (HEK) cells (CRL1573) were

from American Tissue Culture Collection (Manassas, VA, USA).

Minimum essential media (MEM), fetal bovine serum (FBS),

phosphate-buffered saline (PBS), gentamicin, trypsin, HEPES buffer,

acrylamide and N,N¢-methylenebisacrylamide were obtained fromCanadian Life Technologies (Burlington, Ontario, Canada).

Receptor constructs

The HA-tagged truncated D1A dopaminergic receptors (Fig. 1)

were constructed using a PCR-based methodology and the rat HA-

tagged D1A receptor as template. Briefly, a stop codon following

residues Asp425 (Asp425Stop; D425), Gly379 (Gly379Stop; D379)or Cys351 (Cys351Stop; D351) was introduced using PCR primersas detailed elsewhere (Tiberi et al. 1996; Chaar et al. 2001).

HA-tagged wild-type (WT) and truncation receptor constructs were

subcloned in the pCMV5 expression vector.

684 A. Jackson et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 683–697

Cell culture and transfection

Human embryonic kidney 293 (HEK) cells were grown in MEM

containing Earle’s salts, heat-inactivated FBS [10% (v/v)] and

gentamicin (10 lg/mL) at 37�C in a 5% CO2 environment. Cellsseeded in 100-mm dishes (2.5 · 106 cells/dish) were transientlytransfected by a modified calcium-phosphate method (Didsbury

et al. 1991) using a total amount of 5 lg receptor DNA/dish. Whenless than 5 lg DNA of receptor constructs was employed to

transfect cells, empty pCMV5 vector was used to normalize the total

amount of DNA (5 lg). Experiments were done with cells from 34to 55 passages. After an overnight incubation with the DNA-calcium

phosphate precipitate, HEK cells were washed with PBS, trypsi-

nized, reseeded in 100-mm dishes (� 3 · 106 cells) and 6- or 12-well plates (� 7.5 · 105 and 3 · 105 cells per well, respectively)and grown for an additional 36–40 h.

Membrane preparation and radioligand binding

Membrane preparation and radioligand binding studies were

performed essentially as described (Iwasiow et al. 1999; Chaar

et al. 2001). Transfected HEK cells were washed with PBS, scraped

in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4; 5 mM EDTA), and

centrifuged twice at 40 000 g for 20 min at 4�C. The pellet wasresuspended in binding buffer (50 mM Tris-HCl, pH 7.4; 120 mM

NaCl; 5 mM KCl, 4 mM MgCl2; 1.5 mM CaCl2; 1 mM EDTA) using

a Brinkmann Polytron (17 000 r.p.m. for 15 s). Binding assays were

performed with 100 lL of membranes in a total volume of 500 lLusing [N-methyl-3H]-SCH23390 as radioligand. In the present study,

the total receptor number was measured using membranes incu-

bated with a saturating concentration of [N-methyl-3H]SCH23390

(� 5 nM) in the presence or absence of 10 lM flupentixol (todelineate the non-specific binding) for 90 min at 25�C. The bindingassays were stopped using rapid filtration through glass fiber filters

(GF/C, Whatman) and the filters were washed three times with 5 mL

of cold washing buffer (50 mM Tris-HCl, pH 7.4; 120 mM NaCl).

The bound radioactivity was calculated by liquid scintillation

counting using a Beckman Counter (LS1701). Protein concentra-

tions were measured using the Bio-Rad Laboratories (Ontario,

Canada) assay kit with bovine serum albumin (BSA) as standard.

Whole cell phosphorylation

Whole cell phosphorylation experiments were performed in HEK

cells transfected with HA-tagged WT or truncated D1A receptors

using the following amounts of DNA per dish: 0.03 lg of WT,0.015 lg of D425, 0.05 lg of D379, or 5 lg of D351. Under thesetransfection conditions, the total functional receptor numbers for

WT and truncated receptors were similar. Following the overnight

incubation with the DNA–calcium phosphate precipitate, the

transfection medium was removed and replaced with fresh MEM.

Fig. 1 Schematic representation of the amino acid sequence of wild-

type rat D1A dopaminergic receptor. Truncation sites (scissors) and

potential intracellular Ser/Thr phosphorylation sites (filled circles) are

represented.

Dopamine receptor phosphorylation and endocytosis 685


A day prior to the phosphorylation assay, cells were reseeded in six-

well dishes with �1 · 106 cells/well and grown for an additional18 h. Cells were then put in 20 mM HEPES-buffered phosphate free

MEM (pH 7.4) containing gentamicin (10 lg/mL) and 0.2 mCi/mL[32P]orthophosphate and labeled for 90 min at 37�C. At the end ofthe labeling period, cells were incubated in the presence of 0.1 mM

ascorbic acid (control) or 10 lM DA (treated) for 10 min. At the endof incubation, dishes were put on ice, and cells washed three times

with ice-cold PBS. Cells were solubilized by adding 0.5 mL of

RIPA+ buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM

EDTA, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate,

0.1% (w/v) SDS, 10 mM NaF, and 10 mM disodium pyrophosphate)

containing protease inhibitors (20 lg/mL PMSF; 5 lg/mL aproti-nin; 1 lg/mL pepstatin A; 10 lg/mL benzamidine, leupeptine, andsoybean trypsin inhibitor). Solubilized cell extracts were transferred

to 1.5 mL conical tubes and each well washed with 0.5 mL of

RIPA+ buffer. The cell extracts (1 mL) were solubilized for an

additional hour at 4�C using a rotating wheel. Supernatants wereclarified by centrifugation at 15 000 g for 15 min at 4�C andtransferred to new tubes. Two aliquots (15 lL) of the supernatantfractions were taken for protein assay using BSA as standard (Bio-

Rad DC protein assay kit). The protein concentration of the different

solubilized receptor preparations was essentially similar. Superna-

tant fractions were precleared by adding 50 lL of 10% (v/v) proteinA-Sepharose beads in 2% (w/v) BSA and rotated for 1 h at 4�C, andtransferred to new tubes containing 50 lL of rat monoclonal anti-HA affinity matrix. After overnight incubation at 4�C, anti-HAaffinity matrix was pelleted, and the supernatant discarded. The

beads were then washed four times with 1 mL of ice-cold ristocetin-

induced platelet agglutination RIPA+ buffer and dried. Subse-

quently, 60 lL of SDS sample buffer (25 mM Tris-HCl (pH 6.5),8% (v/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol) was

added to each tube and immunocomplexes were dissociated at room

temperature for 2 h. Immunocomplexes were resolved by SDS–

polyacrylamide gel electrophoresis using 10% gels. Gels were dried

and exposed to Kodak Biomax MR Films (Mandel Scientific,

Montreal, Quebec, Canada) at )80�C overnight. The extent ofreceptor phosphorylation was quantified with Typhoon PhosphorI-

mager 8600 (Amersham Pharmacia Biotech) and values normalized

for lane background and receptor number, and expressed as fold

relative to basal phosphorylation of the WT receptor.

Electrophoretic mobility of wild-type and truncated D1A

dopaminergic receptors

We used a sequential immunoprecipitation and immunoblotting

approach to assess the electrophoretic mobility of the WT and

truncated D1A receptors in HEK cells. Briefly, HEK cells trans-

fected with 5 lg DNA of empty pCMV5, HA-tagged WT ortruncated D1A receptors were seeded and grown in six-well dishes

as described above. Dishes were put on ice and for each transfection

condition, cells from two wells were solubilized with 0.5 mL of

RIPA+ buffer, pooled and subjected to immunoprecipitation

essentially as described in the previous section. Immunocomplexes

were incubated in 60 lL of SDS sample buffer and resolved bySDS–polyacrylamide gel electrophoresis using 10% gels. Proteins

were then transferred to a 0.45-lm nitrocellulose membrane (MicronSeparations Inc., MA, USA), incubated sequentially with primary

mouse monoclonal anti-HA antibodies (1 : 750 of 0.4 lg/mL stock)

and secondary mouse HRP-linked F(ab¢)2 fragment (1 : 5000), andimmobilized HA-tagged receptors were detected by chemilumines-

cence using ECL reagents.

Whole cell cAMP assay and receptor desensitization

The ability of WT and truncated D1A receptors to stimulate AC

activity under control and DA pretreatment conditions was assessed

using whole cell cAMP assays as described previously (Iwasiow

et al. 1999; Chaar et al. 2001). HEK cells transfected with

HA-tagged WT or truncated receptors (WT, 0.01 lg DNA/dish;D425, 0.005 lg DNA/dish; D379, 0.02 lg DNA/dish; D351,0.05 lg DNA/dish) were seeded in 12-well dishes and incubatedovernight in fresh MEM medium supplemented with 5% (v/v) FBS,

gentamicin (10 lg/mL), and [3H]adenine (2 lCi/mL; 24 Ci/mmol)at 37�C in a 5% CO2 environment. To assess receptor desensitiza-tion, cells were incubated in labeling medium with 0.1 mM ascorbic

acid only (control) or in the presence of 10 lM DA (treated) for5 min at 37�C. At the end of the treatment, the medium was

aspirated and each well washed twice with 2 mL of PBS. Cells were

then incubated in 1 mL of 20 mM HEPES-buffered MEM contain-

ing 1 mM IBMX with increasing concentrations of DA (in the

presence of 0.1 mM ascorbic acid) for 10 min at 37�C. At the end ofthe incubation period, the medium was removed, and each well

filled with 1 mL of lysis solution containing 2.5% (v/v) perchloric

acid, 1 mM cAMP, and [14C]cAMP (3.75 nCi, �7500 cpm) for30 min at 4�C. The lysates were then transferred to tubes containing0.1 mL of a neutralizing solution (4.2 M KOH), and precipitates

were pelleted by a low-speed centrifugation (�500 g) at 4�C. Theamount of intracellular [3H]cAMP was measured from supernatants

purified by sequential chromatography using Dowex and alumina

columns as described (Johnson et al. 1994). The amount of

[3H]cAMP (CA) over the total amount of intracellular [3H]adenine

(TU) was computed to determine the relative AC activity and

expressed as CA/TU · 1000. The basal value obtained under eachexperimental condition was subtracted, and the net intracellular

cAMP produced with each DA concentration was expressed as

percentage of the D1A receptor-mediated maximal activation of AC

under control conditions. Dose–response curves to DA were

analyzed by a four-parameter logistic equation using ALLFIT

(DeLean et al. 1978).

Immunocytochemistry

HEK cells were transiently transfected with WT (0.03 lg DNA/dish), D425 (0.015 lg DNA/dish), D379 (0.05 lg DNA/dish) orD351 (5 lg DNA/dish) receptor, and seeded on 12-mm diametercoverslips in a 24-well dish. The total number of functional WT and

truncated receptors obtained under these transfection conditions

were essentially similar (�5 pmol/mg membrane protein). Cells oncoverslips were incubated with 0.1 mM ascorbic acid only (control)

or in the presence of 10 lM DA (treated) for 30 min at 37�C. At theend of incubation, coverslips were washed briefly with PBS at room

temperature. Cells were then fixed with 4% paraformaldehyde in

PBS for 20 min at room temperature and washed in PBS. Coverslips

were incubated with 0.37% glycine and 0.27% ammonium chloride

in PBS for 10 min twice, blocked with 1% BSA/0.1% saponin/PBS

for 30 min at room temperature, removed from the well and placed

on parafilm in a humidified dish. Excess blocking solution was

decanted and coverslips incubated with mouse monoclonal HA



antibody (1 : 300 of 0.4 lg/lL stock) in 1% BSA/0.1% saponin/PBS for 1 h at room temperature (100 lL per coverslip). Cellswere washed with 1% BSA/0.1% saponin/PBS for 5 min (five

times), incubated in secondary antibody [Alexa-488 goat anti-mouse

(1 : 500)] in 1% BSA/0.1% saponin/PBS for 30 min in dark at room

temperature followed by three 10-min washes. Coverslips were

incubated in �SlowFade Equilibrating Buffer� for 5 min, andexcess solution decanted prior to mounting coverslips with �Slow-Fade antifade reagent� in glycerol buffer (Molecular Probes). Fornon-permeabilized conditions, saponin was omitted during the

experimental procedure. Confocal laser microscopy (Bio-Rad MRC-

1024MP) was utilized to visualize immunofluorescence and capture

images.

Statistical analysis

Data are reported as arithmetic means ± SE unless stated otherwise.

Homogeneity of variances was determined prior to the statistical

treatment of data as described (Sokal and Rohlf 1981). One-sample

t-test and analysis of variance (one-way ANOVA) with Newman–

Keuls’ multiple comparison test were used to compare two or

multiple groups, respectively. The statistical tests were done using

GraphPad PRISM version 3.00 for Windows (GraphPad Software, San

Diego CA, USA, http://www.graphpad.com). Statistical analyses

were performed with a level of significance established at p < 0.05.

Results

Three sequentially truncated forms (D425, D379, and D351)of the rat HA-tagged D1A receptor were utilized to explore

the role specific CT regions play in regulating DA-promoted

phosphorylation, desensitization and endocytosis of this

heptahelical D1-like subtype (Fig. 1). In our recent study,

we have shown that these truncation receptors bind to

[N-methyl-3H]SCH23390 with high affinity and express at

high but different total receptor numbers in HEK cells

suggesting a role for the CT of D1A receptor in the ligand-

independent regulation of its cell surface expression (Chaar

et al. 2001). Additionally, we have demonstrated that shorter

truncation receptors display a significant increase in DA

affinity (D379 and D351) and agonist-independent activity(D351) suggesting that structural determinants located

between Cys351 and Asp425 restrain the D1A receptor

conformation (Chaar et al. 2001). The ability of D425 andD379 truncation receptors to activate maximally AC in HEKcells incubated in the presence of 10 lM DA remains

unchanged when compared with WT (Chaar et al. 2001). In

striking contrast, however, DA-mediated maximal activation

of AC in D351-expressing cells is augmented significantly incomparison with WT and longer truncation receptors (Chaar

et al. 2001). Thus, removal of CT sequences downstream of

Cys351 increases somewhat the ability of heptahelical D1A

receptors to stimulate maximally the AC activity in HEK

cells. These results suggest that D351 receptor is potentiallyrefractory to cellular processes partaking in agonist-mediated

regulation of heptahelical GPCR responsiveness. In a series

of experiments described in the next sections, we have tested

this hypothesis.

Sequential truncation of the cytoplasmic tail delineates

two regions regulating agonist-promoted phosphorylation

and desensitization of the D1A receptor

In a series of experiments, WT-, D425-, D379- and D351-expressing HEK cells were solubilized and immunoprecip-

itated as described in �Materials and methods� to assess theelectrophoretic mobility of the truncation receptors. Our

results indicate that WT migrates as a broad band of about

75–85 kDa in HEK cells (Fig. 2a) as shown previously

(Tiberi et al. 1996). The HA-tagged D425, D379 and D351proteins run as a single broad band ranging from 65 to

80 kDa while no broad band was detected in mock-

transfected cells (Fig. 2a). Under the experimental conditions

used, the D351 immunoreactivity signal was lower (Fig. 2a),a finding consistent with our previous studies showing that in

HEK cells D351 displays a total functional receptor numbersignificantly lower than WT-, D425- and D379 (Chaar et al.2001). We then tested the ability of the truncation receptors

to undergo DA-induced phosphorylation in cells expressing

similar total functional receptor numbers (Fig. 2b). WT

receptors display a three-fold increase above basal in agonist-

mediated phosphorylation following a 10-min DA exposure

(Figs 2b and c). Sequential truncation of the CT leads to a

significant and drastic reduction in agonist-induced D1A

receptor phosphorylation. Indeed, D425 and D379 display35% and 90% decrease in agonist-dependent receptor phos-

phorylation, respectively (Figs 2b and c). The DA-promoted

D351 phosphorylation was not detectable (Figs 2b and c).Thus, phosphorylation sites and/or structural determinants

found in CT regions bordered by Gly379 and Asp425 as well

as Asp425 and Thr446 regulate the extent of agonist-

mediated D1A receptor phosphorylation.

In agreement with the phosphorylation data, our desen-

sitization studies indicate that removal of putative phos-

phorylation sites on CT correlates with a significant loss

in DA-mediated desensitization of D1A receptors. A pre-

exposure of HEK expressing WT receptors to 10 lM DA(5 min) leads to a 3.5-fold rightward shift in EC50 and

25% decrease in maximal activation of AC (Fig. 3). A

deletion of the last 21 amino acids of CT (D425) reducesDA-induced D1A receptor desensitization as reflected by

the 2-fold rightward shift in EC50 value (Fig. 3). The loss of

DA-mediated maximal activation of AC elicited by D425under desensitized conditions remains unchanged in com-

parison with desensitized WT receptors (Fig. 3). Moreover,

DA-mediated D1A subtype desensitization was further

reduced in cells expressing D379 and D351, two longertruncation receptors (Figs 1 and 3). Indeed, under our desen-

sitization conditions, D379 (lacking 67 residues) and D351(lacking 95 residues) display no rightward shift in the EC50



values when compared with their respective controls

(p > 0.05) whereas the loss in the extent of DA-mediated

maximal activation of AC accounts for �20% (Fig. 3). Thesedata suggest that removal of CT sequences delimited by

Gly379 and Asp425, and Asp425 and Thr446, leads to a

significant attenuation in the extent of DA-promoted D1A

receptor desensitization as indexed by the rightward shift in

EC50 values and loss of maximal activation of AC. Overall,

our results provide evidence for a functional role of two

structurally different CT regions in mediating DA-induced

D1A receptor phosphorylation and desensitization.

Deletion of cytoplasmic tail sequences delimited

by Cys351 and Gly379 defines a region involved

in DA-mediated endocytosis of the D1A receptor

In a series of experiments using permeabilized HEK cells, we

asked whether the CT plays a functional role in rapid agonist-

mediated D1A receptor endocytosis. Confocal images of

immunofluorescence localization were obtained using per-

meabilized HEK cells expressing similar total functional

receptor numbers of WT and various truncation mutants.

Unstimulated HEK cells expressing WT, D425 or D379exhibit an immunostaining mainly localized on cell surface

(Fig. 4). Permeabilized HEK cells expressing WT, D425 orD379 exhibit a punctate perinuclear labeling in the cytoplasmassociated with a loss of cell surface immunofluorescence

following a 30-min incubation with 10 lM DA (Fig. 4). Instriking contrast, monoclonal HA antibody staining of

permeabilized and unstimulated HEK cells expressing D351reveals that D351 is localized to both the cell surface andcytoplasm, which remains essentially unchanged following

DA treatment (Fig. 4). The strong intracellular staining

observed in unstimulated D351-expressing cells, however,impedes the accurate determination of DA-promoted D351endocytosis. Therefore, to gain further information on the

agonist-promoted endocytosis of D351 receptors, we per-formed additional experiments using non-permeabilized cells

expressing either WTor D351 to assess the loss of cell surfaceimmunofluorescence in the presence or absence of DA. Using

this approach, we observed that a majority of WT-expressing

cells exhibit a reduction of cell surface immunostaining

following DA exposure while D351-expressing cells displayno significant change in the level of immunofluorescence

detected on cell surface under similar experimental conditions

(Fig. 5). Thus, our data support the view that CT sequences

located between Cys351 and Gly379 play an important role in

agonist-promoted D1A receptor endocytosis.

Cytoplasmic tail sequences of the D1A receptor

differentially regulate the rate of DA-mediated

AC activation

To explore further the functional role of D1A receptor

phosphorylation/desensitization and endocytosis/resensitiza-

tion in regulating Gs coupling to AC stimulation, we

(c)

(b)

(a)

Fig. 2 Phosphorylation of the wild-type and truncated D1A receptors

expressed in human embryonic kidney (HEK) cells. (a) Sequential

immunoprecipitation and immunoblotting of wild-type (WT) and trun-

cation receptors expressed in HEK cells as described under �Materials

and methods�. Shown is a representative example of an experiment

repeated two times. The receptor number in pmol/mg of membrane

protein (expressed as the arithmetic mean ± SE) was 21.9 ± 3.00

(WT), 32.7 ± 2.15 (D425), 20.8 ± 1.15 (D379) and 6.45 ± 0.35 (D351).

(b) HEK cells transfected with empty pCMV5 vector or the HA-tagged

WT, D425, D379 or D351 receptor were treated with or without 10 lM

dopamine (DA) for 10 min and subjected to immunoprecipitation using

anti-HA affinity matrix as described under �Experimental Procedures�.

Immunocomplexes were then resolved by SDS–polyacrylamide gel

electrophoresis using 10% gels, and receptor phosphorylation visual-

ized by autoradiography at )80�C overnight. Shown is a representa-

tive example of an experiment repeated three times. (c) Receptor

phosphorylation described in (b) was quantified with Typhoon Phos-

phorImager 8600 normalized relative to basal phosphorylation of WT.

Results are expressed as arithmetic means ± SE of three experi-

ments. The receptor number in picomoles/mg of membrane protein

(expressed as the arithmetic mean ± SE) was 6.33 ± 1.21 (WT),

6.30 ± 1.13 (D425), 6.47 ± 0.50 (D379) and 9.97 ± 0.57 (D351).

*p < 0.05 when compared with WT basal, #p < 0.05 when compared

with WT dopamine, Yp < 0.05 when compared with D425 dopamine.

WT, wild-type; DA, dopamine.



performed time-course studies to determine the observed rate

constant (kobs) for activation of AC by WT and different

truncated receptors in intact cells (Fig. 6a). Computational

and experimental studies suggest that phosphorylation,

desensitization, endocytosis and resensitization regulate sign-

aling efficacy of GPCRs (Su et al. 1976; Lauffenburger and

Linderman 1993; Riccobene et al. 1999; Ferguson 2001). We

reasoned that these cellular processes could also modulate

D1A receptor-mediated kobs for AC activation. Our time-

course studies indicate that deletion of CTsequences influence

differentially the activation rate of AC in HEK cells (Fig. 6b).

Indeed, kobs values measured for D425 (0.088 min)1) andD379 (0.124 min)1) increase incrementally and are statisti-cally different in comparison with WT (0.061 min)1). Intrigu-

ingly, further deletions of CTsequences, as indexed using time

course studies with D351, lead to a kobs value (0.059 min)1)

that is indistinguishable from WT (Fig. 6b). Overall, intrin-

sic differences in agonist-mediated receptor desensitization,

endocytosis and potentially resensitization thereafter, may

explain the kobs values measured in HEK cells expressing WT

and truncation mutants.

Discussion

Previous studies have used a truncation approach to investi-

gate the functional relationship between the CT of GPCRs

and agonist-induced desensitization, phosphorylation and

endocytosis (Krupnick and Benovic 1998). Regardless of

reports showing functional effects of numerous truncation

receptors, it has been difficult to put forward common

generalities with reference to the role of the CT or specific

regions thereof (e.g. distal versus proximal regions) in

Fig. 3 Dopamine-induced desensitization of adenylyl cyclase activity

in human embryonic kidney (HEK) cells transfected with wild-type

(WT) and truncated D1A receptors. HEK cells transfected with

HA-tagged WT, D425, D379 or D351 were seeded in 12-well dishes

and labeled with [3H]adenine as described under �Materials and

methods�. Cells were treated with or without 10 lM dopamine (DA) for

5 min at 37�C. At the end of the treatment, each well was washed

twice with PBS, and cells were incubated in the presence or absence

of increasing concentrations of DA for 10 min at 37�C. Results are

expressed as arithmetic means ± SE of two to six experiments done in

triplicate determinations. Each point was first expressed as percent-

age of maximal response obtained under control experimental condi-

tions. Production of intracellular cAMP is plotted as a function of log of

DA concentrations, and curves were analyzed by simultaneous curve

fitting using ALLFIT and GraphPad PRISM version 3.00. The insets show

the EC50 rightward shift values (treated/control ± approximate SE as

obtained with ALLFIT). Statistical significance was determined using

unconstrained and constrained simultaneous curve fitting. When the

maximal response was constrained to share a common value, �control�

and �treated� conditions were statistically different (p < 0.05) in all

cases. The receptor number in pmol/mg of membrane protein

(expressed as the arithmetic mean ± SE) was 1.10 ± 0.06 (WT, con-

trol), 1.04 ± 0.08 (WT, treated), 1.90 ± 0.40 (D425, control),

1.55 ± 0.35 (D425, treated), 1.65 ± 0.05 (D379; control), 1.45 ± 0.25

(D379; treated), 1.88 ± 0.26 (D351; control) and 1.95 ± 0.13 (D351;

treated). *p < 0.05 when compared with control. WT, wild-type.



regulating agonist-induced GPCR phosphorylation, desensi-

tization and endocytosis. For instance, the CT of histamine

H2 receptors is dispensable for agonist-induced desensitiza-

tion but essential for agonist-mediated receptor endocytosis

(Fukushima et al. 1997). In an opposite fashion, the CT of

b2-adrenergic receptors is crucial for agonist-promotedphosphorylation and desensitization but not critical for

endocytosis (Strader et al. 1987; Bouvier et al. 1988).

Meanwhile the CT is important for epinephrine-induced

phosphorylation, desensitization and endocytosis of a1B-adrenergic receptors (Lattion et al. 1994). The low degree of

identity within the primary structure of the CT of these

GPCRs as well as the cellular models used to study the

truncated receptor properties could explain the reported

�functional discrepancies�. These studies are just few exam-ples highlighting the functional complexity of the CT struc-

tural determinants involved in agonist-induced regulation of

GPCR responsiveness. Overall, these studies implicate that

the functional effects of CT mutations or truncations are

GPCR- and/or cell-specific.

Another important point, however, is that very few studies

have used a combination of phosphorylation, desensitization

and endocytosis assays to assess the molecular interplay of

these post-translational processes (Krupnick and Benovic

1998; Ferguson 2001). In the present study, our truncation

strategy removed 18 out of 20 potential Ser/Thr phosphory-

lation sites distributed along the CTof the D1A receptor using

clusters of six residues (Fig. 1). Data obtained with these

truncation receptors strongly suggest that a deletion of phos-

phorylation sites clustered within two specific CT regions

(one being delimited by Gly379 and Asp425 and the other by

Asp425 and Thr446) inhibits almost completely DA-induced

receptor phosphorylation. Concomitant to this inhibition, we

also observed a loss of D1A receptor desensitization in

DA-treated cells expressing D425 and D379 as indexed by theEC50 rightward shift and maximal activation of AC values. In

a similar fashion to D425, truncation of the last 18 amino acidsof the nucleotide P2Y2 GPCR leads to a loss of UTP-induced

desensitization (Garrad et al. 1998). However, in the latter

study, the role of UTP-induced phosphorylation in desensi-

tization of truncated P2Y2 receptors was not documented

(Garrad et al. 1998). Interestingly, removal of distal CT

sequences of D1A receptors leads to functional effects that are

different from those observed with similar deletions in other

GPCRs. Indeed, a 49-amino acid truncation (deletion of six

Ser/Thr residues) of cannabinoid CB1 receptors did not alter

agonist-induced desensitization (Jin et al. 1999). Moreover,

a1B-adrenergic receptors harboring a 46-amino acid trunca-tion (deletion of 15 Ser/Thr residues) display no changes in

agonist-induced phosphorylation and desensitization in COS-

7 cells (Lattion et al. 1994; Diviani et al. 1997). Thus, distal

CT sequences seem to play a distinct role in agonist-induced

phosphorylation and desensitization of GPCRs. Importantly,

GRK phosphorylation sites have been mapped to distal CT

regions of some GPCRs (Krupnick and Benovic 1998; Pitcher

et al. 1998). Studies have shown that HEK cells express

endogenously moderate levels of GRK2/3/6 and b-arrestin1/2(Menard et al. 1997; Hall et al. 1999b). Hence, GRK2,

GRK3 and/or GRK6 may represent the potential kinases

involved in DA-dependent D1A receptor phosphorylation,

and suggest the existence of GRK2/3/6 phosphorylation sites

located downstream of Gly379. A coexpression of various

members of GRK family in HEK cells has shown that the

BASAL

WT

∆425

∆379

∆351

10 µM DA

Fig. 4 Intracellular sorting of wild-type and truncated D1A receptors

expressed in human embryonic kidney (HEK) cells in the presence or

absence of dopamine. HEK cells transfected with HA-tagged WT,

D425, D379 or D351 were seeded on coverslips in a 24-well dish and

treated with or without 10 lM dopamine (DA) for 30 min at 37�C. Cells

were then permeabilized and fixed as described under �Materials and

methods�. Confocal laser microscopy was used to visualize immuno-

fluorescence and capture images. Confocal images were collected

with a 40 · )1.3 NA oil immersion lens and the settings for iris, gain

and background levels were identical for each image. Shown is a

representative example of an experiment repeated three times. The

total functional receptor number was not altered following DA treat-

ment (data not shown). WT, wild-type.



agonist-occupied D1A receptor is a substrate for GRK2,

GRK3 and GRK5 (Tiberi et al. 1996) but not GRK6 (R. M.

Iwasiow and M. Tiberi, unpublished data). Thus, the loss of

agonist-induced phosphorylation observed in D425 and D379-expressing cells may reflect the removal of potential GRK2

and/or GRK3 phosphorylation sites. In addition, the cAMP-

dependent kinase (PKA) has been implicated in the D1A

receptor phosphorylation and desensitization (Zhou et al.

1991; Zamanillo et al. 1995; Jiang and Sibley 1999; Gardner

et al. 2001). Studies have also shown that PKA and GRKs can

regulate GPCR phosphorylation and desensitization in a

synergistic fashion (Moffett et al. 2001). Interestingly, the

residue Ser380, previously identified as a potential PKA site

using a D1A-CT fusion protein (Zamanillo et al. 1995) was

deleted in D379 mutant. As a result, the drastic decreaseobserved in DA-mediated phosphorylation of D379 in com-parison with WT and D425 is potentially explained by thePKA site deletion.

Our studies also show that DA-induced desensitization

was significantly attenuated in D351-expressing cells as

compared with WT- and D425-expressing cells (Fig. 2). Webelieve that the attenuation of DA-induced D351 desensi-tization (as indexed by the lack of EC50 rightward shift in

DA-treated cells) is not linked to the higher constitutive

activation of D351 in HEK cells (Chaar et al. 2001). In fact,an interesting finding of the present study is, in spite of a

complete loss of DA-promoted receptor phosphorylation,

D351 can still undergo DA-induced desensitization asindexed by the reduction in the maximal activation of AC

in DA-treated cells. Importantly, D351 display desensitiza-tion features similar to those of D379, a longer truncationreceptor that does not display an increase in its constitutive

activation (Chaar et al. 2001). Moreover, D379 undergoesDA-induced phosphorylation, albeit to a significant lesser

extent than WT and D425 (Fig. 2). Although we cannot ruleout the possibility that Ser/Thr residues located upstream

Cys351 and/or on intracellular loops are also phosphoryl-

ated but could not be detected under our experimental

conditions, our results obtained with D379 and D351suggest that a GPCR phosphorylation-independent process

BASAL

WT

∆351

10 µM DA

Fig. 5 Plasma membrane sorting of wild-type (WT) and D351 recep-

tors expressed in human embryonic kidney (HEK) cells in the presence

or absence of dopamine. HEK cells transfected with HA-tagged WT

or D351 were seeded on coverslips in a 24-well dish and treated with

or without 10 lM dopamine (DA) for 30 min at 37�C. Non-permeabilized

cells were fixed as described under �Materials and methods�. Confo-

cal laser microscopy was used to visualize immunofluorescence

and capture images. Confocal images were collected with a 40 · )1.3

NA oil immersion lens and the settings for iris, gain and background

levels were identical for each image. Shown is a representative

example of an experiment repeated two times. The total functional

receptor number was not altered following DA treatment (data not

shown).



potentially regulates agonist-induced desensitization of D1A

receptors. Our findings are somewhat different from previ-

ous studies using truncated b2- and a1B-adrenergic recep-tors (Bouvier et al. 1988; Lattion et al. 1994). Indeed, these

studies have shown that a deletion of the CT eliminates

completely the rapid agonist-induced phosphorylation and

desensitization of b2- and a1B-adrenergic receptors (Bou-vier et al. 1988; Lattion et al. 1994). Meanwhile, a trunca-

tion of the CT of chemokine CXC receptor 4 (CXCR4) or

neurokinin-2 receptor leads to a total abrogation of the rapid

agonist-induced phosphorylation of these GPCRs while

retaining their ability to undergo agonist-induced desensiti-

zation (Alblas et al. 1995; Haribabu et al. 1997). Interest-

ingly, the agonist-induced desensitization of the truncated

CXCR4 was linked to the agonist-mediated protein kinase C

phosphorylation of a downstream signaling intermediate of

CXCR4, the phospholipase C b3 isoform (Haribabu et al.1997). However, the potential role for an agonist-mediated

phosphorylation of downstream signaling intermediates in

DA-induced D1A receptor desensitization requires further

studies.

It is well established that GRK-mediated phosphorylation

increases receptor binding affinity for arrestins, a key

biochemical process underlying the extent of receptor desen-

sitization (Krupnick and Benovic 1998). Studies have also

shown that arrestins can interact with sites on intracellular

regions of WT and truncated receptors (Ferguson et al. 1996;

Wu et al. 1997; Krupnick and Benovic 1998). Therefore, it is

possible that removal of CT sequences exposes arrestin

binding sites on intracellular regions of the D1A receptor and

may explain the small DA-mediated desensitization observed

in cells expressing D379 and D351. Recently, a study hasshown that b-arrestin1 conjugated to a green fluorescentprotein was recruited from the cytosol to plasma membrane

following DA exposure of HEK cells expressing rat D1A

receptors (Barak et al. 1997). This study suggests that

arrestins can bind to and regulate D1A receptor signaling

properties. Data obtained in our lab suggest that overexpres-

sion of arrestin isoforms can modulate the D1A receptor

responsiveness (data not shown). Alternatively, binding of

intracellular factors to cytoplasmic regions of the D1A

subtype may be involved in a phosphorylation- and arrestin-

independent desensitization of the receptor. Notwithstanding

the potential role of a GPCR phosphorylation-independ-

ent process in agonist-induced D1A receptor desensitiza-

tion, our study implies that the loss of D1A subtype

responsiveness following DA treatment is linked somewhat

to the phosphorylation of specific residues located on distal

CT regions. Our results also suggest that, in addition to

promoting desensitization, DA-induced phosphorylation may

potentially regulate other functional properties of the hepta-

helical D1A receptor as shown for other GPCR types (Menard

et al. 1997; Cao et al. 1999; Hall et al. 1999a; Walker et al.

1999).

Fig. 6 Time course of dopamine-mediated stimulation of adenylyl

cyclase activity in human embryonic kidney (HEK) cells transfected

with wild-type (WT) and truncated D1A receptors. (a) Adenylyl cyclase

activity was assessed in single wells of a six-well dish in the presence

of 10 lM dopamine for various time periods using whole cell cAMP

assays as described under �Materials and methods�. Results are

expressed as arithmetic means ± SE of two to three experiments done

in triplicate determinations. Production of intracellular cAMP is plotted

as a function of time, and curves for wild-type (dashed line) and

truncated receptors (solid line) were best fitted to one–phase expo-

nential association equation [Y ¼ Ymax(1 ) e–kX)] using GraphPad

PRISM version 3.00. For each experimental condition, results were

normalized as percentage of the Ymax value derived from non-linear

regression analyses of curves depicted in bottom graph. The Ymax

values were 20.1 ± 8.56 (WT), 17.7 ± 3.58 (D425), 17.5 ± 4.05 (D379)

and 46.8 ± 9.36 (D351). The receptor number in picomoles/mg of

membrane protein (expressed as the arithmetic mean ± SE) was

2.50 ± 1.20 (WT; 0.02 lg of DNA/dish), 2.33 ± 0.61 (D425; 0.01 lg of

DNA/dish), 2.23 ± 0.66 (D379; 0.02 lg of DNA/dish) and 2.10 ± 0.36

(D351; 0.25 lg of DNA/dish). (b) Best-fit values of apparent first order

rate constants (kobs) expressed in min)1 ± approximate SE are shown.

Statistical significance was determined using unconstrained and con-

strained curve fitting. *p < 0.05 when compared with WT, #p < 0.05

when compared with D425, Yp < 0.05 when compared with D379. CA/

TU, [3H]cAMP formed divided by the total uptake. WT, wild-type.



Indeed, an additional important aspect of our studies is

the potential role phosphorylation may play in DA-promo-

ted D1A receptor endocytosis. Studies have suggested that

GRK-mediated phosphorylation and arrestin binding parti-

cipate in agonist-promoted GPCR endocytosis (Krupnick

and Benovic 1998; Pierce and Lefkowitz 2001). Further-

more, a recent study has shown that the agonist-promoted

secretin receptor endocytosis is unaltered by overexpression

of GRK isoforms or dominant negative b-arrestin1 mutantsbut blocked by PKA inhibition (Walker et al. 1999).

Biochemical and immunofluorescence data obtained with

WT and truncation receptors suggest that removal of

phosphorylation of residues located on the most distal CT

region (delimited by Asp425 and Thr446 residues) do not

abolish DA-induced D1A receptor endocytosis. In a dis-

tinguishing manner, removal of distal CT sequences of

cannabinoid CB1 or angiotensin II type 1 A receptor elimi-

nates agonist-induced receptor endocytosis (Thomas et al.

1995a; Thomas et al. 1995b; Smith et al. 1998; Jin et al.

1999). Our findings are also somehow in disagreement with

a recent study showing that mutation of Ser431, Thr439 and

Thr446 leads to an abrogation of agonist-induced D1A

receptor internalization while being without any significant

effect on agonist-mediated receptor desensitization (Lamey

et al. 2002). Most importantly, our immunofluorescence and

whole cell phosphorylation data imply that the lack of

detectable DA-induced receptor phosphorylation in D351-expressing cells may explain, at least partially, impairment

in DA-promoted D351 endocytosis (Figs 4 and 5). Indeed,in contrast to D351, D379 retains its ability to endocytosefollowing DA exposure, an observation that is potentially

explained by the phosphorylation of residues located

between Cys351 and Gly379. Thus, phosphorylation of

residues located within the CT region enclosed by Cys351

and Gly379 may be important for agonist-induced receptor

endocytosis while playing a limited role in receptor

desensitization. Furthermore, our studies may point to the

existence of endocytic motifs localized between Cys351 and

Gly379 required for agonist-promoted D1A receptor endo-

cytosis. Alternatively, we cannot rule out the presence of

sequences acting as inhibitory constraints of agonist-

promoted receptor endocytosis. In fact, studies using serially

truncated parathyroid hormone, parathyroid hormone-related

protein and somatostatin receptors have suggested that

positive and negative endocytotic signals located on the CT

govern the agonist-induced GPCR endocytosis (Huang

et al. 1995; Hukovic et al. 1998). Further work is required

to explore the role of these CT regions in regulating the

kinetics and extent of DA-promoted D1A receptor endo-

cytosis as removal of CT sequences has been shown

to slow agonist-induced endocytosis of several GPCRs

(Lattion et al. 1994; Huang et al. 1995; Thomas et al.

1995a; Garrad et al. 1998; Hukovic et al. 1998; Smith et al.

1998).

Agonist-promoted desensitization and endocytosis have

been shown to play an important role in the modulation of

ligand efficacy, receptor-G protein coupling status and

resensitization (Riccobene et al. 1999; Pierce and Lefkowitz

2001). Moreover, experimental and mathematical studies

suggest that kobs for effector enzyme activation (e.g. AC) is

dependent on the total available receptor number and/or

receptor coupling status but independent of total effector

activity and G protein levels (Su et al. 1976; Lauffenburger

and Linderman 1993; Riccobene et al. 1999). As depicted in

our model (Fig. 7a), phosphorylation and desensitization of

heptahelical D1A receptors would lead to a decrease

(negative effect) in kobs for AC activation following DA

stimulation. Meanwhile, endocytosis and resensitization

would modulate an increase (positive effect) in kobs. The

increased kobs values determined in cells expressing D425 andD379 are in agreement with a diminution in agonist-promotedphosphorylation/desensitization (receptor-G protein coupling

status) of these truncated receptors (Figs 7b and c). Data

obtained with D351 denote, however, that the kobs valuedepends on additional cellular processes, notably endocyto-

sis and resensitization (Fig. 7d). Indeed, our time-course

experiments suggest that the decreased kobs detected in D351-expressing cells, which exhibit similar desensitization fea-

tures of D379-expressing cells, may be potentially explainedby impairment in the agonist-induced D351 endocytosis(Figs 4 and 5). Thus, impairment in agonist-induced endo-

cytosis may lead to a longer retention time of desensitized

D351 receptors at the plasma membrane in comparison withdesensitized WT and D379 receptors. This assertion impliesthat despite a greater extent in agonist-promoted phosphory-

lation and desensitization, the WT receptor may reestablish its

signaling properties following DA activation faster than

D351, by virtue of a more efficient endocytosis and resen-sitization process. It is worth noting that the effectiveness of

GPCR resensitization is also dependent on specific CT

sequences (Ferguson 2001). Thus, an impaired DA-induced

D351 endocytosis concomitant to D351 ability to undergoagonist-promoted desensitization, even though small, would

then contribute to modulate negatively kobs for AC activation

in comparison with kobs values assessed in cells expressing

D425 or D379 (Fig. 7). Furthermore, impairment of D351endocytosis (Fig. 7d) may expand the half-life of a functional

receptor/G protein/effector complex, and provide an explan-

ation for the significant increase of the extent of DA-mediated

maximal activation of AC in D351-expressing cells (Chaaret al. 2001). Alternatively, as we have previously proposed,

the increase in the extent of agonist-mediated maximal

activation of AC by D351 may also be explained by arestricting mobility of Gsa relative to the truncated receptoras shown for b2-adrenergic receptors (Wenzel-Seifert et al.1998; Chaar et al. 2001).

In conclusion, we have delineated distinct regions within

the CT of the heptahelical D1A receptor that differentially



regulate the rapid agonist-promoted phosphorylation, desen-

sitization and endocytosis of this dopaminergic GPCR.

Furthermore, our study provides evidence for a potential

role of a phosphorylation-independent process in mediating

the short-term agonist-induced desensitization of D1A dop-

aminergic receptors. Importantly, our results may be of

functional significance as the agonist-induced D1A receptor

endocytosis in vivo has recently been demonstrated (Dumar-

tin et al. 1998). Moreover, an internalization process has

been shown to control the availability of heptahelical D1A

receptor at the cell surface in animal models of hyperdop-

aminergia (Dumartin et al. 2000). Our results may then

(a)

(b)

(c)

(d)

Fig. 7 Model depicting the role of GPCR regulatory processes in

controlling dopamine-induced heptahelical D1A receptor activation of

adenylyl cyclase (AC). In this model, the extent and overall observed

rate constant (kobs) for AC activation following dopamine (DA)-induced

activation of D1A receptor as shown in (a) depends on its G protein

coupling status. Agonist-induced GPCR phosphorylation and desen-

sitization exert a negative effect on the receptor-G protein coupling

status. Meanwhile endocytosis and resensitization exert a positive

effect on the receptor-G protein coupling status by allowing the rees-

tablishment of the D1A receptor ability to signal to AC. The summation

of these effects dictates the D1A receptor-mediated kobs value (filled

block arrow) for AC activation in a given cellular system. In cellular

systems harboring D1A receptors that exhibit an incremental loss in

agonist-induced GPCR phosphorylation and desensitization as shown

in (b) (D425) and (c) (D379), the kobs for AC activation would be

increased accordingly. However, in cellular systems expressing D1A

receptors that display also a reduction in agonist-induced GPCR

endocytosis and resensitization thereafter as depicted in (d) (D351),

the kobs for AC activation would be decreased significantly in com-

parison with cells expressing D425 and D379. Furthermore, the

decreased agonist-induced endocytosis of D351 would be retained a

longer time at the plasma membrane and lead to an augmentation in

the extent of DA-mediated maximal activation of AC (hatched block

arrow). See Discussion for details.



prove to be of clinical importance given that the regulation of

D1-like receptor responsiveness is compromised in several

CNS and peripheral pathophysiological disorders (Roseboom

and Gnegy 1989; Okubo et al. 1997; Sanada et al. 1999;

Zhen et al. 2001). It is hoped that a characterization of the

molecular mechanisms regulating the functionality of the

D1A receptor may provide information suggesting novel

therapeutic approaches for conditions displaying a compro-

mised D1A receptor function.

Acknowledgements

We thank Dr Andrew Ridsdale for assistance with confocal micro-

scopy. An operating grant from the Canadian Institutes of Health

Research (CIHR) supported this work (to MT). AJ is a NeuroScience

Canada Foundation/CIHR Fellow and holds a Canadian Psychiatric

Research Foundation Award. RMI and ZYC are recipients of a K. M.

Hunter Doctoral Research Award from CIHR.

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