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Originally published In Press as doi:10.1074/jbc.M401923200 on May 24, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35671-35678, August 20, 2004
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Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal*

Samuel P. Lee{ddagger}§, Christopher H. So{ddagger}§, Asim J. Rashid{ddagger}||, George Varghese{ddagger}, Regina Cheng{ddagger}, A. José Lança{ddagger}, Brian F. O'Dowd{ddagger}, and Susan R. George{ddagger}¶**{ddagger}{ddagger}

From the Departments of {ddagger}Pharmacology and **Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada and the Centre for Addiction and Mental Health, Toronto, Ontario M5T 1R8, Canada

Received for publication, February 22, 2004 , and in revised form, May 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the Gq pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The dopamine D1 receptor has been shown to signal via Gs and Golf proteins to stimulate adenylyl cyclase (1), and there have been reports of calcium signaling by D1 receptor-mediated phosphatidylinositol hydrolysis by phospholipase C (PLC),1 presumably through Gq coupling. Although PLC activity was not detected in studies on the D1 receptor expressed in COS7 cells (2) or in Chinese hamster ovary and baby hamster kidney cells (3), it has been shown that activation of the D1 receptor in brain tissue (4, 5) or in Xenopus oocytes injected with mRNA taken from striatum (6) resulted in phosphatidylinositol turnover. This discrepancy suggests the involvement of a factor in D1 receptor-mediated PLC activity that is present in native tissues but absent in heterologous expression systems. An examination of the above studies where PLC activation was observed indicates that, although D1 receptor-selective antagonists blocked phosphatidylinositol turnover demonstrating the involvement of the D1 receptor, the amount of agonist used to elicit the response was greater than 100 µM, a concentration at which even selective ligands may bind other receptors. For example, SKF 81297, a highly D1-selective agonist, has a Ki for the D1 receptor of ~1 nM but has a Ki for the D2 receptor of ~900 nM (7). This analysis suggests the possibility that the D1 agonist-mediated PLC stimulation by high concentrations of drugs observed in physiological systems may have resulted from the coincident activation of another receptor.

There are considerable data indicating that the dopamine D1 and D2 receptors are functionally linked. For example, behavioral studies have shown that co-stimulation of the D1 receptor is essential for D2 agonists to produce maximal locomotor stimulation (8) and that activation of both D1 and D2 receptors was required to augment the acute effects of cocaine action (9). Biochemical and electrophysiological evidence has also supported D1-D2 receptor synergism. Combined administration of specific D1 and D2 agonists potentiated immediate early gene expression (10, 11). Also, long term depression of synaptic transmission after dopamine depletion could be restored by dopamine or co-administration of specific D1 and D2 agonists but not by either selective agonist alone (12). Although the mechanism for the D1-D2 receptor interaction in these studies showing functional linkage is not clearly understood, there is evidence that an interaction between the receptors occurring within the same cell may be involved in some instances. For example, synergistic potentiation of the D2 receptor-mediated enhancement of arachidonic acid release by co-administration of D1-selective and D2-selective agonists in cells expressing both receptors has been observed (13). Furthermore, it has been shown that D1 subclass and D2 subclass receptors are co-expressed in neurons of the rat striatum (14), suggesting the possibility that there may be a direct D1-D2 receptor association.

Therefore, we postulated that dopaminergic PLC-mediated calcium signaling results from the co-activation of dopamine D1 and D2 receptors and the formation of a novel signaling unit represents the molecular basis for the functional interaction between these receptors. In investigating this hypothesis, we demonstrated that co-activation of co-expressed D1 and D2 receptors resulted in a PLC-mediated increase of intracellular calcium levels, a signaling pathway not activated when either one of the receptors was singly activated. Further, we also found evidence that D1 and D2 receptors associate within neurons and thereby potentially form a novel signaling complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Receptor Expression—All cell culture and transfection reagents were obtained from Invitrogen. COS7 and HEK293T cells were maintained as monolayer cultures at 37 °C in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. Transient expression was performed using LipofectAMINETM with DNA encoding human D1 receptor or the long isoform of the human dopamine D2 receptor that had been inserted into pcDNA3 vector (Invitrogen). For immunoprecipitation experiments, HA and FLAG epitope tags were introduced after the initiation methionine of the D1 and D2 receptors, respectively, by PCR. Stable cell lines co-expressing the N terminus HA epitope-tagged D1 receptor and N terminus FLAG epitope-tagged human D2 receptor were created in HEK293TSA cells utilizing the pBudCE 4.1 vector (Invitrogen). Briefly, the D1 receptor cDNA was inserted into the EF1{alpha} multicloning site and the D2 receptor cDNA into the cytomegalovirus site. Antibiotic-resistant clones (selected with 200 µg/ml zeocin) of each transfection were isolated and tested for expression of corresponding receptors using saturation binding analysis.

Measurement of Calcium Signal—Calcium mobilization assays were carried out using a FLEXstation multiwell plate fluorometer (Molecular Devices, Sunnyvale, CA). Stably transfected cells were seeded in black microtiter plates at a density of ~105 cells/well and grown for 24 h. The cells were then loaded with 2 µM Fluo-4AM indicator dye (Molecular Probes, Inc., Eugene, OR) in growth medium supplemented with 20 mM HEPES and 2.5 mM probenecid for 1 h and subsequently washed twice with Hanks' balanced salt solution without sodium bicarbonate and phenol red (Invitrogen). Base-line fluorescence values were measured for 15 s, and changes in fluorescence corresponding to alterations in intracellular calcium levels upon the addition of agonists thereafter were also recorded. Fluorescence values were collected at 3-s intervals for 150 s. For calculation of dose-response curves, the peak fluorescence values for each agonist concentration were determined and analyzed using Prism software (GraphPad, San Diego, CA). In signaling pathway inhibition studies, cells were treated with 10 µM H89 (Calbiochem), 10 µM U73122 [GenBank] (Sigma), 1 µM wortmannin (Calbiochem), or 500 nM bisindolamide I (Calbiochem) for 1 h or with 0.125–1 µg/ml pertussis toxin (Sigma) for 18 h prior to the calcium measurements.

Adenylyl Cyclase Activity—Adenylyl cyclase assays were conducted essentially as described previously (15). The assay mix contained 0.02 ml of membrane suspension (10–25 µg of protein), 0.012 mM ATP, 0.1 mM cAMP, 0.053 mM GTP, 2.7 mM phosphoenolpyruvate, 0.2 units of pyruvate kinase, 1 unit of myokinase, and 0.13 µCi of [33P]ATP in a final volume of 0.05 ml. For the inhibition experiments, 1 µM forskolin was included in the assay mix. The mixture was incubated with 10–3 to 10–12 M SKF 81297 or quinpirole for 20 min, and enzyme activities were determined. Data were analyzed by computer-fitted nonlinear least-squares regression.

Generation of Striatal Cultures—Neuronal cultures derived from the rodent striatum were generated using standard protocols. Briefly, striatal tissue was dissected from newborn rats (P1) and washed with calcium- and magnesium-free HEPES-buffered saline. The tissue was then incubated in a solution consisting of 0.25% trypsin and 0.5% DNase I for 20 min at 37 °C, followed by three washes in calcium-free HEPES-buffered saline with 12 mM magnesium sulfate. Cells were dissociated by trituration in a solution containing calcium-free HEPES-buffered saline, 12 mM magnesium sulfate, and 0.5% DNase I and then centrifuged. This was followed by resuspension in plating medium (Dulbecco's minimal essential medium with 2 mM glutamine and 10% fetal bovine serum). Cells were plated on poly-L-lysine and laminin-coated 12-mm coverslips in 24-well plates at a density of ~105 cells/coverslip. The following day, the plating medium was replaced with serum-free Neurobasal A medium (Invitrogen) supplemented with glutamine and B27 supplement (Invitrogen). Half the medium was replaced with fresh medium every 2–3 days. Immunocytochemical analysis of neurons was performed after 7–10 days in vitro.

Fluorescence Immunohistochemistry of Brain Tissue—Human brain tissue was obtained from the Brain and Tissue Bank for Developmental Disorders at the Department of Pediatrics, University of Maryland. Brain tissue from male Sprague-Dawley rats was also examined. Immunocytochemical procedures followed the indirect (secondary antibody-labeled) fluorescent technique described previously (16). The primary antibody for the D1 receptor was obtained from Sigma (catalog no. D-187), and the anti-D2 receptor antibody was acquired from Chemicon (Temecula, CA; catalog no. AB5084P). Secondary antibodies to visualize D1 and D2 receptors were labeled with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate (TRITC) (Molecular Probes), respectively. Confocal laser microscopy was performed using a Zeiss LSM 510 system. Primary antibody specificity was tested using immunofluorescence microscopy of HEK 293T cells individually expressing each of the five dopamine receptor subtypes. Secondary antibody specificity was tested by control experiments in which no primary antibody was used.

Immunocytochemistry of Cultured Neurons—Neurons were washed twice in phosphate-buffered saline, fixed in 4% paraformaldehyde for 30 min at 37 °C, washed again in phosphate-buffered saline, and then blocked for 2 h at room temperature in 10% goat serum, 0.1% Triton X-100, 0.1 mg/ml bovine serum albumin in phosphate-buffered saline. Primary antibodies were added to each well in a 1:10 dilution of the blocking solution, and cells were incubated overnight at 4 °C. The next day, cells were washed and incubated with secondary antibody for 1 h at room temperature. Following another series of washes, the coverslips were mounted on slides. The primary antibodies used in the staining were mouse monoclonal anti-D1 receptor (Chemicon catalog no. MAB5290) and rabbit anti-D2 receptor (Chemicon catalog no. AB5084P).

Demonstration of Dopamine Receptor Antibody Specificity—Expression vector constructs encoding N-terminal c-Myc epitope-tagged human D3 dopamine receptor or encoding N-terminal HA epitope-tagged human D5 dopamine receptor were generated via PCR and insertion into pcDNA3 vector. A FLAG epitope-tagged human D4.4 dopamine receptor cDNA construct was generously provided by Dr. Hubert Van Tol (University of Toronto and the Centre for Addiction and Mental Health, Toronto, Canada). HEK 293T cells were singly transfected with one of these constructs or with an expression vector encoding either the HA-tagged D1 receptor or the FLAG-tagged D2 receptor described earlier. The cells were fixed on glass coverslips in a manner similar to the one outlined above for cultured neurons. The receptors expressed in these cells were then probed by immunohistochemistry using the D1 or D2 receptor antibodies from Sigma (catalog no. D-187) and Chemicon (catalog no. AB5084P), respectively, and using an antibody corresponding to the epitope tag of the receptor.

Immunoprecipitation—Immunoprecipitation from HEK293T cells was performed as described previously (17), except the 3F10 anti-HA (Roche Applied Science) and 9E10 anti-c-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies were used for detection of the HA and c-Myc epitope, respectively. For immunoprecipitation from brain, striatal tissue was dissected from adult female Sprague-Dawley rats, and the procedure was carried out using a protocol described previously (18). Striatal membranes (500 µg) were solubilized and incubated with 5 µg of rabbit anti-rat D1 (Chemicon catalog no. AB1765P) or rabbit anti-rat D2 (Chemicon catalog no. AB5084P). Eluted protein was resolved by electrophoresis and then transferred onto polyvinylidene difluoride membranes for Western blot analysis. Immunoblots were probed using a monoclonal D1 receptor antibody (Sigma catalog no. D-187).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Co-activation of D1 and D2 Receptors Elevates Intracellular Calcium—We investigated whether the co-activation of dopamine D1 and D2 receptors resulted in a calcium signal. When cells co-expressing D1 and D2 receptors were stimulated by concurrent administration of equal concentrations of SKF 81297, a D1-selective agonist, and the D2-selective agonist quinpirole, a significant increase in intracellular calcium levels was observed (Fig. 1, A and B). Calcium mobilization was initially detected ~2–5 s following agonist application, and the signal peaked within 30 s of the initial exposure to agonist (Fig. 1A). This maximal response was dose-dependent, requiring a 65.4 ± 12.9 nM concentration of each of the selective agonists to generate 50% of the maximal response (Fig. 1B).



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  		FIG. 1.
Calcium signal is observed only when the D1 and D2 receptors are co-activated. A, time course of the fluorescence measurements detected by the FLEXstation multiwell plate fluorometer corresponding to increases in intracellular Ca2+ levels in cells co-expressing D1 and D2 receptors following simultaneous activation by both SKF 81297 and quinpirole. A.F.U., arbitrary fluorescence units. The arrow indicates the time point when the drugs are added. The curves shown are representative of the five replicate measurements performed in four independent experiments. B–D, dose-response curves showing calcium signal in cells co-expressing the D1 and D2 receptors (B) or expressing either the D1 receptor alone (C) or the D2 receptor alone (D) and treated with SKF 81297, quinpirole, or both SKF 81297 and quinpirole. The data used to plot each curve were from at least three independent experiments. E, fluorescence measurements corresponding to increases in intracellular Ca2+ levels in cells co-expressing D1 and D2 receptors following simultaneous activation by both SKF 81297 and quinpirole (10 µM each) or by 10 µM ATP. The arrows point to the peak responses of the P2Y1 and P2Y2 purinergic receptors and of the co-activated D1 and D2 receptors. The curves shown are representative of the five replicate measurements performed in 3–5 independent experiments. F, fluorescence measurements corresponding to increases in intracellular Ca2+ levels show that in cells expressing both D1 and D2 receptors, the calcium response elicited by 10 µM each of SKF 81297 and quinpirole could be significantly attenuated by the addition of either 1 µM eticlopride or 1 µM SCH 23390.

 
Treatment of the cells co-expressing the D1 and D2 receptors with either SKF 81297 or quinpirole individually did not increase intracellular calcium levels (Fig. 1B). No significant change in intracellular calcium levels could be observed in cells expressing only the D1 receptor that were stimulated with SKF 81297 or quinpirole or with both agonists concurrently (Fig. 1C). Similarly, there was no apparent calcium signal induced by SKF 81297, quinpirole, or the co-administration of both drugs in cells expressing only the D2 receptor (Fig. 1D).

For comparison of the temporal characteristics of the calcium signal, activation of P2Y purinergic receptors was compared with that of the D1 and D2 receptors. HEK 293 cells have been shown to endogenously express G protein-coupled P2Y1 and P2Y2 purinergic receptors that stimulate PLC via Gq coupling when activated (19). Interestingly, the rate of calcium signal propagation is distinct for these two receptors, since the P2Y2 receptor-mediated signal occurs at a faster rate than the signal mediated by P2Y1 receptors (20). The agonist-induced increase in intracellular calcium-dependent fluorescence by the D1 and D2 receptor co-activation was temporally similar to that detected for Gq-coupled P2Y1 purinergic receptors activated by ATP (Fig. 1E).

To confirm the requirement for concurrent activation of both D1 and D2 receptors in order to evoke calcium signal in cells expressing both D1 and D2 receptors, cells co-expressing D1 and D2 receptors were treated with either SCH 23390, a D1-selective antagonist, alone, or with eticlopride, a D2-selective antagonist, alone. Notably, the dual agonist-induced calcium signal was attenuated by the addition of SCH 23390 or by the addition of eticlopride (Fig. 1F).

We hypothesized that dopamine treatment would elicit calcium increase in a manner similar to that arising by concurrent administration of SKF 81297 and quinpirole. No change in intracellular calcium levels was generated by dopamine in cells expressing only D1 or D2 dopamine receptors; however, when cells co-expressing D1 and D2 receptors were incubated with dopamine, an increase in intracellular calcium levels was seen (Fig. 2A). Again, the agonist-induced intracellular calcium increase could be blocked by the addition of SCH 23390 or eticlopride (Fig. 2B).



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  		FIG. 2.
Dopamine co-activation of co-expressed D1 and D2 receptors evokes calcium signal, but dopamine activation of D1 receptor or D2 receptor alone does not. A, dose-response curves of fluorescence measurements detected by the FLEXstation multiwell plate fluorometer corresponding to increases in intracellular Ca2+ levels in cells co-expressing the D1 and D2 receptors or expressing either the D1 receptor alone or the D2 receptor alone and treated with dopamine. A.F.U., arbitrary fluorescence units. B, the response elicited by 10 µM dopamine in cells co-expressing both receptors could be significantly attenuated by the addition of either 1 µM eticlopride or 1 µM SCH 23390.

 
In order to examine whether coupling to the adenylyl cyclase signaling pathway was affected by co-expression of D1 and D2 receptors, we examined cells co-expressing D1 and D2 receptors stimulated with SKF 81297 or with quinpirole (Fig. 3). The ability of the D1 receptor to stimulate adenylyl cyclase and of the D2 receptor to inhibit adenylyl cyclase was unaffected. There was no statistically significant difference in the maximum stimulation of adenylyl cyclase and the EC50 between the D1-D2 receptor-expressing cells and cells expressing only the D1 receptor. The same was true for the maximum inhibition of adenylyl cylcase and the EC50 between the D1-D2 receptor-expressing cells and cells expressing only the D2 receptor. These observations suggest that cells expressing both receptors may be capable of evoking three distinct dopamine-mediated signals simultaneously.



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  		FIG. 3.
Adenylyl cyclase stimulation by the D1 receptor and adenylyl cyclase inhibition by the D2 receptor is not altered in cell co-expressing both receptors. Dose-response curves showing adenylyl cyclase stimulation or inhibition in cells expressing D1 receptor alone, D2 receptor alone, or co-expressing D1 and D2 receptors. Membranes from the cells were activated with increasing concentrations of SKF 81297 (A) or were treated with forskolin and activated by increasing concentrations of quinpirole (B).

 
Signal Transduction Pathway for D1-D2 Receptor Co-activation—In order to determine the mechanism of the calcium signal generation, we attempted to block key components of several signal transduction pathways while co-stimulating the receptors with both D1- and D2-selective agonists.

Gs protein activation by the D1 receptor has been implicated in calcium signaling via protein kinase A (PKA)-mediated mechanisms (21–23). Therefore, cells expressing D1 and D2 receptors were treated with H89, an inhibitor of PKA, and co-activated by D1 and D2 receptor agonists. There was no effect of H89 on the increase in intracellular calcium levels, suggesting that co-activated D1 and D2 receptor-mediated calcium signaling was not dependent on PKA (Fig. 4A).



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  		FIG. 4.
The calcium signal induced by D1 and D2 receptor co-activation is abolished with PLC inhibitor but not other kinase inhibitors or Gi/Go inactivation. A, intracellular Ca2+ levels measured by a FLEXstation scanning fluorometer. Cells stably expressing both D1 and D2 receptors were pretreated with H89, U73122 [GenBank] , wortmannin, bisindolamide I, or pertussis toxin as outlined under "Experimental Procedures." The cells were then stimulated with a 1 µM concentration of both SKF 81297 and quinpirole. The asterisk (p < 0.02) and double asterisk (p < 0.03) represent statistically significant differences from the untreated measurement. The results shown are from three independent experiments, in each of which there were five replicates. A.F.U., arbitrary fluorescence units. B, fluorescence measurements corresponding to intracellular Ca2+ levels in cells co-expressing D1 and D2 receptors or left untransfected. The cells were treated with U73122 [GenBank] and stimulated with both SKF 81297 and quinpirole (10 µM each) or by 10 µM ATP to activate endogenously expressing purinergic receptors. A statistically significant decrease in Ca2+ levels following U73122 [GenBank] treatment was seen for both D1-D2 receptor signaling (p < 0.0001) and purinergic receptor signaling (p < 0.0007). The results shown are from three independent experiments, in each of which there were five replicates. C, effect of pertussis toxin on the Ca2+ signal mediated by SKF 81297 and quinpirole (10 µM each) or by 10 µM ATP. Cells co-expressing the D1 and D2 receptors were treated with several concentrations of PTX and then stimulated by agonists. The results shown are representative of one of three independent experiments, in each of which there were five replicates.

 
We then examined whether simultaneously activated D1 and D2 receptors increase intracellular calcium levels via a PLC-dependent pathway. Cells were treated with U73122 [GenBank] , a PLC inhibitor. Notably, the increase in intracellular calcium levels by D1 and D2 receptor co-activation was markedly attenuated (>90%) after treatment with U73122 [GenBank] (Fig. 4A). U73122 [GenBank] also inhibited the calcium signal mediated by the endogenous, Gq-linked P2Y receptors (Fig. 4B), indicating that the inhibitor was blocking PLC. These results suggest that, when co-activated, D1 and D2 receptors may couple to Gq and signal through an inositol phosphate pathway to cause the release of intracellular calcium.

G protein-coupled receptor stimulation of PLC may also occur via a protein kinase C (PKC)-mediated mechanism (24). Furthermore, there is evidence that lipid products of phosphoinositide 3-kinase action may generate a PLC-mediated calcium signal (25). We therefore investigated whether PKC or phosphoinositide 3-kinase played a role in generating the calcium signal evoked by co-activated D1 and D2 receptors. When cells co-expressing D1 and D2 receptors were treated with bisindolamide I, a PKC inhibitor, and co-stimulated with D1 and D2 agonists, no attenuation of calcium signal was observed (Fig. 4A), suggesting that PLC was not stimulated by the actions of PKC. Similarly, when we examined the effects of the phosphoinositide 3-kinase inhibitor, wortmannin, on the D1-D2 receptor-mediated calcium signal, no significant changes were detected, suggesting no role for phosphoinositide 3-kinase in the novel signal generated by D1-D2 receptor coactivation (Fig. 4A).

Although the G{alpha}i/o subunits, the G proteins traditionally associated with D2 receptor signal transduction, have not been directly implicated in increases of intracellular calcium levels, the pertussis toxin (PTX)-sensitive signaling pathways have been linked to the activation of calcium signals. In such pathways, G{beta}{gamma} dimers of PTX-sensitive G proteins have been shown to stimulate PLC directly (reviewed in Ref. 26) or by modulating the Gq pathway, which in turn activates PLC (27). We investigated whether the co-activation of D1 and D2 receptors resulted in calcium signaling via a PTX-sensitive pathway. Treatment of cells co-expressing the D1 and D2 receptors with PTX resulted in a partial reduction (~25–35%) in the maximum calcium stimulation caused by co-administration of SKF 81297 and quinpirole (Fig. 4, A and C). This effect did not appear to be dependent on the concentration of PTX, since the level of inhibition was virtually identical for all doses of PTX used. Notably, we have shown that 0.125 µg/ml PTX completely attenuates D2-mediated adenylyl cyclase inhibition,2 but concentrations of PTX up to 1 µg/ml did not completely block D1-D2 receptor-mediated calcium signal. In our positive control involving endogenous Gq-linked P2Y receptors, P2Y receptor-mediated calcium signal in HEK 293 cells was also partially blocked (~25–30%) by PTX (Fig. 4C). These observations suggest that PTX may diminish the Gq-mediated calcium signal by co-activated D1-D2 receptors in a nonspecific manner but that the Gi/o pathway is not directly involved in the generation of the signal.

Pharmacological Characterization of Co-expressed D1 and D2 Receptors—In order to determine whether the co-expression of D1 and D2 receptors resulted in a change in the conformation of the binding pocket of either receptor, ligand binding studies were performed. Competition of selective radiolabeled antagonist binding by dopamine or selective agonists indicated no significant change in binding affinity when D1 and D2 dopamine receptors were co-expressed (data not shown). In saturation binding studies, there was no change in the affinity (Kd) of the antagonists [3H]SCH 23390 or [3H]raclopride for the D1 and D2 receptors, respectively, upon co-expression. The Bmax and Kd values for [3H]SCH 23390 binding were 2797 ± 14 fmol/mg protein and 0.59 ± 0.02 nM for the cells stably expressing the D1 receptor alone and were 2071 ± 60 fmol/mg protein and 0.59 ± 0.02 nM for cells stably expressing both D1 and D2 receptors. The Bmax and Kd values for [3H]raclopride binding were 1105.5 ± 27.5 fmol/mg protein and 0.97 ± 0.084 nM for D2 receptor-expressing cells and 2449 ± 90.5 fmol/mg, and 0.675 ± 0.155 nM for cells stably expressing D1 and D2 receptors. These results suggest that co-expression did not affect the ligand binding pocket of the D1 or the D2 receptors.

D1 and D2 Receptors Are Colocalized in Neurons—In order to determine whether there was a physiological basis for the novel response by D1 and D2 receptor co-activation, we examined whether these two receptors were co-expressed and co-localized within the same neurons. The expression of D1 and D2 receptors within neurons in brain tissues and cultured neurons was analyzed. D1-like and D2-like receptors have been shown to be co-localized in neurons of the rodent striatum (14); however, there has been some controversy as to whether the D1 and D2 receptor subtypes are co-expressed in the same cell (14, 28). Using antibodies that were specific for the D1 and D2 receptors, we substantiated the subtype selectivity and performed immunohistochemical analysis of human and rat brain. In the caudate nucleus, prominent immunolabel was observed for both D1 and D2 receptors in cell bodies of individual medium spiny neurons (Fig. 5A). Distinct puncta corresponding to immunolabel for both receptors could be observed, which overlapped significantly in cells expressing both D1 and D2 receptors.



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  		FIG. 5.
D1 and D2 receptors are co-expressed and co-localized in human and rat brain. Confocal microscopy of the immunocytochemical visualization of D1 and D2 receptors in human caudate (A) and rat frontal cortex (layer V) (B) and in rat striatal cultures (C). The D1 antibody labeling was visualized by secondary antibody conjugated to fluorescein isothiocyanate (green), and the D2 antibody labeling was visualized by secondary antibody conjugated to TRITC (red), shown separately and as an overlay. In the D1 and D2 co-expressing rat brain neuron in B, processes containing predominantly D1 receptor or D2 receptor are indicated by the arrows.

 
In the rat frontal cortex (Fig. 5B), pyramidal cells were shown to co-express both D1 and D2 receptors, whereas interneurons appearing to only express D2 receptors were observed. Within cortical pyramidal neurons, immunolabel for both receptors was co-localized in neurites up to at least 40 µm away from the cell body. Neuronal processes were also observed originating from the same neuron that contained either exclusively D1 receptor immunolabel or D2 receptor immunolabel (Fig. 5B, arrows), suggesting the possibility of at least three distinct populations of dopamine receptors within the same cell.

In neuronal cultures derived from neonatal rat striatum, D1 and D2 receptors were co-expressed in a significant proportion of cells (Fig. 5C). D2 receptor immunostaining was observed in every neuron examined, with intense immunolabel both in the cell bodies and throughout the full extent of their dendrites. Expression of D1 receptors was distinct from that of D2 receptors in that staining was observed in only 40–60% of neurons, and, in most cases, immunolabeling was restricted to the cell body and proximal dendrites.

The high degree of specificity of the antibodies used for D1 and D2 receptor detection was confirmed by immunocytochemical staining of cells heterologously expressing each of the five dopamine receptor subtypes individually (Fig. 6). Epitope tagging of the receptors and subsequent detection with antibodies directed against the tags were used to confirm receptor expression in the cells, and the receptor-directed antibody was used to show specificity and lack of cross-reactivity. The D1 receptor-specific antibody only stained cells expressing the D1 receptor and none of the cells expressing the other four dopamine receptors. Similarly, no immunofluorescence was detected using the D2 receptor-specific antibody in cells other than those expressing the D2 receptor. Therefore, our immunocytochemical analyses of the rat brain, both slices and cultured neurons, and of the human brain slices show that there are distinct, but overlapping patterns of D1 and D2 receptor expression. This observation indicates that these two receptors are co-expressed within the same neurons and have the potential to interact with each other.



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  		FIG. 6.
Specificity of D1 and D2 receptor antibodies. HEK 293 cells expressing epitope-tagged dopamine receptors D1–D5 were incubated with the D1 receptor and D2 receptor antibodies and an antibody directed against the epitope. The D1 antibody labeling (column 1) and the epitope-recognizing antibodies in column 4 were visualized by secondary antibody conjugated to fluorescein isothiocyanate (green). The D2 antibody labeling (column 3) and the epitope-recognizing antibodies in column 2 were visualized by secondary antibody conjugated to TRITC (red). Labeling by the antibodies directed against the epitope tags demonstrated that the cells were expressing receptors at the cell surface. The D1 receptor antibody only immunolabeled cells expressing D1 receptor, whereas the D2 receptor only labeled cells expressing D2 receptor.

 
Co-immunoprecipitation of D1 and D2 Receptors—We have demonstrated previously that D1 and D2 receptors exist as homodimers in heterologous expression systems (29, 30) and in human and rat brain tissue (31). Based on the ability of dopamine receptors to oligomerize, we hypothesized that an intermolecular interaction between the D1 and D2 receptors may account for the novel signal observed. HA epitope-tagged D1 receptors and c-Myc epitope-tagged D2 receptors were co-expressed in COS7 cells and immunoprecipitated using anti-HA and anti-c-Myc antibodies (Fig. 7A). When HA antibody precipitates were imunoblotted with a c-Myc probe, a band corresponding to the D2 receptor was immunodetected. Similarly, co-immunoprecipitation of HA-D1 receptor with c-Myc antibody was also observed.



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  		FIG. 7.
Endogenous D1 and D2 receptors are part of the same signaling complex in rat brain. A, co-immunoprecipitation of HA-D1 receptor and c-Myc-D2 receptor. Membranes from cells expressing c-Myc D2 receptor (lane 1) or HA-D1 receptor (lane 3) or co-expressing both receptors (lanes 2 and 4) were immunoprecipitated with anti-HA antibodies and immunodetected using anti-c-Myc antibodies (lanes 1 and 2) or immunoprecipitated with anti-c-Myc antibodies and immunodetected using anti-HA antibodies (lanes 3 and 4). The arrows indicate immunodetected receptor and IgG. B, co-immunoprecipitation of D1 and D2 receptors from rat striatum. Immunodetection of the D1 receptor with monoclonal D1 receptor antibody in a striatal membrane preparation (lane 5) or following immunoprecipitation by the D1 antibody (lane 6) or D2 antibody (lane 7). No signal was observed when striatal protein was omitted (lanes 8 and 9). IP, immunoprecipitation; IB, immunoblot.

 
In blots of immunoprecipitates from rat striatal membranes (Fig. 7B), a ~50-kDa band corresponding to the D1 receptor was detected in proteins purified with a D1 receptor antibody (Fig. 7B, lane 1). When immunoprecipitation for the D2 receptor was performed and the precipitate was probed for the D1 receptor, the D1 receptor was detected (Fig. 7B, lane 2), indicating that D1 and D2 receptor were part of the same protein complex in rat striatum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have provided the first demonstration that two co-expressed G protein-coupled receptors, when co-activated, can form a completely novel signaling complex. We have shown that concurrent agonist activation of co-expressed dopamine D1 and D2 receptors evoked a PLC-mediated elevation of intracellular calcium levels, a cellular response not generally associated with these receptors that have been shown to predominantly signal via the adenylyl cyclase effector system. The activation of a Gq-linked pathway by a complex of D1 and D2 receptors is an exciting prospect, given that these receptors are thought to transduce signal predominantly by coupling to Gs/olf and Gi/o proteins, respectively. The calcium signal was not observed upon stimulation of either receptor expressed alone or upon activation of cells co-expressing D1-D2 receptor with an agonist selective for only one of the receptors. It has been reported that heteromers of the CCR2 and CCR5 chemokine receptors (32) and heteromers of the µ- and {delta}-opioid receptors (17) may couple to G proteins distinct from those associated with homogeneous populations of their constituent receptors. However, the cellular response induced by these heteromers is not different from that of the individual receptors (increased intracellular Ca2+ for the CCR2/CCR5 receptors and inhibition of adenylyl cyclase for the µ/{delta} receptors). Our results represent the first demonstration of the coupling to a novel effector resulting from the formation of a complex involving two distinct G protein-coupled receptors.

Our analyses showed that neurons and regions within neurons were populated by D1 or D2 receptors exclusively and by co-localized, interacting D1 and D2 receptors, and cells that express both receptors were able to mediate both stimulation and inhibition of adenylyl cyclase in addition to generating a calcium signal. Therefore, it is likely that there are at least three different types of dopaminergic signaling units generated by these two receptor subtypes. The nature of a signaling pathway activated by D1 receptors may completely depend on whether or not the D1 receptors are associated with D2 receptors.

The precise mechanism by which Gq couples to the D1-D2 heteromeric complex remains to be determined. The direct interaction of Gq with the D1 receptor upon co-activation with the D2 receptor or Gq coupling to D2 receptor mediated by co-activation with the D1 receptor are both possibilities. Interestingly, there is recent evidence that only one G protein is associated for each receptor homodimer of the BLT1 leukotriene B4 receptor (33). A similar configuration was recently speculated to be the case for rhodopsin, based on crystallography data that indicate that, given the relative sizes of G proteins and rhodopsin, only one G protein could associate with a receptor homodimer due to spatial restrictions (34). These studies suggest a third possibility in which G protein selectivity may be determined by the conformation of both the D1 and D2 receptors.

Indirect calcium signaling by the D1 receptor has been shown to occur in some heterologous expression systems (21–23). This response is attributed to cAMP and PKA, since identical calcium increases can be induced by cAMP analogs and can be blocked by PKA inhibitors. In our study, PKA inhibitor treatment did not attenuate the intracellular calcium increases observed, thus excluding this pathway as a potential mechanism of calcium activation by co-activated D1 and D2 receptors. Cross-talk between Gi- and Gq-coupled receptors mediated by G{beta}{gamma} exchange has been observed to result in potentiation of Gq-mediated signaling due to Gi-coupled receptor activation (35). A cross-talk mechanism does not appear to underlie the novel signaling unit generated by co-activated D1 and D2 receptors, since our observations indicate that neither receptor couples to a Gq pathway in the absence of concurrent activation of both receptors. It has also been demonstrated that a single transmembrane domain protein known as calcyon can interact with the D1 receptor to allow D1 receptor-mediated increases in intracellular calcium levels (36). However, the activation of the calcyon-D1 receptor complex requires "priming" by the prior activation of a co-expressed Gq-coupled receptor, and therefore, calcyon is unlikely to be involved in the calcium signal seen in this study.

It was important to establish a physiological basis for D1 and D2 receptor interaction, since there has been considerable controversy concerning whether or not D1 and D2 receptors are co-localized within the same neurons (14, 28). Aizman et al. (14) have reported that, in the rat striatum, virtually all neurons expressing D1 subclass receptors also express D2 subclass receptors. However, in situ hybridization studies have shown that D1 and D2 receptors do not have completely overlapping distributions in the striatum (37–39). Using highly specific D1 and D2 receptor antibodies and confocal laser microscopy of human caudate, rat striatum, and cultured neurons from rat striatum, we have shown that these two receptors were co-expressed and co-localized within a significant number of neurons.

In conclusion, we have established, using classical inhibitors of various signal transduction mechanisms, that co-activated D1 and D2 receptors can signal via a PLC-mediated calcium pathway, a pathway not activated by either receptor alone. We have also shown D1 and D2 receptors are co-expressed and co-localized within neurons and that these two receptors are part of the same protein complex. Numerous earlier studies have suggested that these receptors were functionally linked. Therefore, making a logical progression from previous studies of the D1 and D2 receptors, we present a new model for signaling by dopamine D1 and D2 receptors in brain, where these two receptors function together in a complex to elevate intracellular calcium when co-activated. The generation of three unique signals from two receptors provides an example of how diversity of receptor function can be achieved from a limited number of receptors. Although these types of observations add a degree of complexity not traditionally considered in models of receptor function, it is becoming increasingly apparent that analysis of the function of individual receptor gene products alone is insufficient and new paradigms are required. Further investigation of the generation of unique functional properties via receptor-receptor interactions may yield a better understanding of behavior and central nervous system disorders mediated by dopamine and other G protein-coupled receptors. For instance, there is a large body of evidence suggesting that dysfunction of calcium signaling may be associated with the etiology of schizophrenia (reviewed in Ref. 40). In future studies, it would be intriguing to investigate whether this calcium signaling by co-activated D1 and D2 receptors is related to schizophrenia and other dopamine-related disorders.


   FOOTNOTES
 
* This work was supported by grants from the National Institute on Drug Abuse and from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These two authors contributed equally to this work. Back

|| Supported by a Fellowship from the Centre for Addiction and Mental Health. Back

{ddagger}{ddagger} Holder of a Canada Research Chair in Molecular Neuroscience. To whom correspondence should be addressed: Rm. 4358, Medical Sciences Bldg., 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-3367; Fax: 416-971-2868; E-mail: s.george{at}utoronto.ca.

1 The abbreviations used are: PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; PTX, pertussis toxin. Back

2 G. Varghese, B. F. O'Dowd, and S. R. George, unpublished observation. Back



   REFERENCES
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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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