Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor.

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.

G-protein-coupled receptors (GPCRs) 1 represent a large family of seven transmembrane-spanning proteins that transduce cellular responses to numerous extracellular signals, including hormones, neurotransmitters, odorants, and light (1,2). Most cells maintain homeostatic control of their responsiveness to signals through regulating the expression and functional activity of their cell surface GPCRs. One widely studied form of GPCR regulation is that of agonist-induced desensitization. In this process, activation of the GPCR by an agonist also triggers a sequence of events that results in the dampening of the receptor-mediated signal. The mechanisms associated with this "homologous" form of desensitization have been most thoroughly investigated for the ␤ 2 -adrenergic receptor-coupled adenylyl cyclase system (3,4), resulting in the following paradigm. Agonist occupancy of the receptor promotes its phosphorylation by a member of the G-protein-coupled receptor kinase (GRK) family leading to the binding of an arrestin-like protein, ultimately resulting in uncoupling of the receptor from its cognate G-protein and decreased functional signaling. The binding of an arrestin-like molecule also promotes internalization of the receptor through clathrin-coated pits into an endosomal compartment where it may be de-phosphorylated and recycled to the cell surface or degraded via a lysosomal pathway. Although in many instances this desensitization paradigm has been shown to be operative for other G-protein-coupled receptors, recent studies have suggested that there may be significant exceptions and variations to this general scheme.
Much less is known about "heterologous" forms of regulation in which activation of one GPCR, or other receptor system(s), may lead to the desensitization of multiple unrelated receptors in the same cell. Evidence to date suggests that heterologous regulation of receptor-mediated signaling pathways frequently involves phosphorylation of various signaling components by second messenger-regulated protein kinases that are activated by the initial signal (4 -7). Typically, phosphorylation of GPCRs by second messenger activated kinases such as protein kinase A (PKA) or C (PKC) has been suggested to dampen the ability of the receptor to couple to G-proteins and elicit a response, although precise mechanistic details are lacking. It is highly likely that heterologous forms of desensitization may play a critical role in fine-tuning GPCR responses in cells such as neurons that may concurrently receive numerous hormonal or neurotransmitter signals.
Dopamine receptors (DARs) are members of the GPCR superfamily and consist of five structurally distinct subtypes (8). These can be divided into two subgroups on the basis of their structure and pharmacological and transductional properties. The first subgroup, termed "D 1 -like" comprises the D 1 and D 5 DARs that stimulate adenylyl cyclase and raise intracellular levels of cAMP. The second DAR subgroup includes the D 2 , D 3 , and D 4 receptors and is termed "D 2 -like." The D 2 -like DARs are coupled to the inhibition of adenylyl cyclase as well as the modulation of potassium and calcium ion channels. As with other GPCRs, DARs are subject to a wide variety of regulatory mechanisms, which can either positively or negatively modulate their expression and functional activity (9).
The mechanisms underlying various forms of DAR regulation have only begun to be elucidated. The greatest information has been derived from studies using the D 1 DAR, which appears to be phosphorylated and internalized in a GRK-dependent fashion upon agonist activation, although some notable differences from the scheme described above exist (10 -15). In contrast, studies of the D 2 DAR have revealed that regulation of this receptor is extremely complex with agonist activation variably resulting in functional desensitization, sensitization, receptor up-or down-regulation, or no effect at all (reviewed in Ref. 9). Given that the D 2 DAR is the primary target for all known antipsychotic drugs (antagonists) and drugs used to treat Parkinson's disease (agonists), more information concerning the regulatory mechanisms for this receptor may lead to improved therapeutics for treating D 2 DAR-related diseases (16,17). Recent information has, in fact, suggested that the D 2 DAR is a substrate for GRKs in cellular expression systems and that GRK-mediated phosphorylation promotes receptor internalization (18,19). In contrast, little information is available concerning phosphorylation and regulation of the D 2 DAR by second messenger-activated protein kinases, although earlier studies have suggested potential roles for PKA and PKC (20 -24). In our present study, we have now investigated phosphorylation of the D 2 DAR by second messenger-activated protein kinases, and we demonstrate that PKC phosphorylates the D 2 DAR on multiple sites within two domains of the third intracellular loop. This PKC-mediated phosphorylation of the D 2 DAR was demonstrated to promote both functional desensitization and internalization of the receptor protein via a ␤-arrestin-and dynamin-dependent pathway.

EXPERIMENTAL PROCEDURES
Materials-HEK293-tsa201 (HEK293T) cells (25)  Plasmids and Mutagenesis-To create an amino-terminal FLAGtagged construct of the rat D 2L DAR (pSF-D 2L ), a synthetic oligonucleotide encoding a signal sequence and an antigenic epitope of the "FLAG" epitope (26) was inserted in-frame at the beginning of the D 2L DAR coding sequence in the pSR-D2L (27). The XhoI/BamHI fragment that contains the 5Ј-noncoding region of D 2L in pSR-D2L was excised and replaced with the signal sequence and the FLAG epitope. Site-directed mutagenesis was performed using the QuikChange® XL kit from Stratagene (La Jolla, CA). Single or multiple point mutations were created to replace serine residues by alanine or threonine residues by valine. All constructs were verified by DNA sequencing prior to use. The M1 muscarinic receptor expression construct was a gift from Jurgen Wess (National Institutes of Health). The following PKC expression constructs were gifts from the respective individuals: PKC␤I and -(Alexandra Newton) and PKC␦ and -(Alex Toker). The dominant negative mutant constructs for dynamin and ␤-arrestin were gifts from Jeffrey Benovic.
Cell Culture and Transfections-HEK293T cells were cultured in DMEM supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 50 units/ml penicillin, 50 g/ml streptomycin, and 10 g/ml gentamycin. Cells were grown at 37°C in 5% CO 2 and 90% humidity. HEK293T cells were transfected using the calcium phosphate precipi-tation method (Clontech). Cells were seeded in 100-or 150-mm plates, and transfection was carried out at ϳ50% confluency according to the manufacturer's instructions. After 18 h of transfection, the transfection media were replaced with fresh media, and the cells were divided for subsequent experiments.
Whole-cell Phosphorylation Assays-Metabolic labeling of cells and subsequent immunoprecipitation of the D 2L DAR was carried out as described previously (10). Briefly, HEK293T cells were transfected with pSF-D 2L using the calcium-phosphate method. One day after transfection, cells were seeded at 1-1.5 ϫ 10 6 per well of a poly(D)-lysine-coated 6-well plate for phosphorylation assay and ϳ2 ϫ 10 6 cells on a 100-mm dish for radioligand binding assay to quantify the level of receptor expression. The next day, the cells were washed with Earle's balanced salt solution (EBSS) and incubated for 1 h in phosphate-free DMEM with 10% fetal calf serum. Media were removed and replaced with 1 ml of fresh media supplemented with 200 Ci/ml [ 32 P]H 3 PO 4 . After 45 min at 37°C, the cells were then challenged with 1 M PMA or various drugs. Cells were then transferred to ice, washed twice with ice-cold EBSS, and solubilized for 1 h at 4°C in 1 ml of solubilization buffer (50 mM HEPES, 1 mM EDTA, 10% glycerol, 1% Triton X-100, pH 7.4, at 4°C) ϩ 150 mM NaCl supplemented with Complete protease inhibitor mixture and phosphatase inhibitors (40 mM sodium pyrophosphate, 50 mM NaF). The samples were cleared by centrifugation in a microcentrifuge, and the protein concentration was determined by bicinchoninic acid protein assay (Pierce). The level of D 2 DAR expression for each transfection was quantified via radioligand binding assays using the cells from the same transfection. After receptor/protein quantification, equal amounts of receptor protein were then transferred to fresh tubes with 40 l of washed M2-agarose and incubated overnight with mixing at 4°C. The samples were then washed once with solubilization buffer and 500 mM NaCl, once with solubilization buffer and 150 mM NaCl, and once with Tris-EDTA, pH 7.4, at 4°C. Samples were then incubated 2ϫ SDS-PAGE loading buffer for 1 h at 37°C before being resolved by 4 -20% Tris-glycine SDS-PAGE. The gels were dried and subjected to autoradiography. After developing, the band intensity was quantitated by LabWorks TM software (UVP Inc., Upland, CA).

Intact Cell [ 3 H]Sulpiride
Binding-HEK293T cells expressing rat D 2L DAR were seeded 1 day after transfection at a density of 2 ϫ 10 5 cells/well in poly(D)-lysine-coated 24-well plates. The following day, cells were incubated in the absence (control) and presence of 1 M PMA in DMEM for 2 h. Stimulation was terminated by quickly cooling the plates on ice and washing the cells three times with ice-cold EBSS. Cells were then incubated with 0.5 ml of [ 3 H]sulpiride in EBSS (final concentration, 6.4 nM) at 4°C for 3 h 30 min. For saturation binding assays, cells were incubated with 0.2-30 nM [ 3 H]sulpiride. Nonspecific binding was determined in the presence of 5 M (ϩ)-butaclamol. Cells were washed three times with ice-cold EBSS, and 0.5 ml of 1% Triton X-100 was added. Samples were mixed with 5 ml of liquid scintillation mixture and counted with a Beckman LS6500 scintillation counter.

Membrane [ 3 H]Methylspiperone
Binding-HEK293T cells were harvested by incubation with 5 mM EDTA in EBSS and collected by centrifugation at 300 ϫ g for 10 min. The cells were resuspended in lysis buffer (5 mM Tris, pH 7.4, at 4°C; 5 mM MgCl 2 ) and were disrupted using a Dounce homogenizer followed by centrifugation at 34,000 ϫ g for 10 min. The resulting membrane pellet was resuspended in binding buffer (50 mM Tris, pH 7.4). The membrane suspension was then added to assay tubes containing [ 3 H]methylspiperone in a final volume of 1.0 ml. (ϩ)-Butaclamol was added at the final concentration of 3 M to determine nonspecific binding. The assay tubes were incubated at room temperature for 1.5 h, and the reaction was terminated by rapid filtration through GF/C filters pretreated with 0.3% polyethyleneimine. Radioactivity bound to the filters was quantitated by liquid scintillation spectroscopy.
Determination of cAMP Production-HEK293T cells were seeded into poly(D)-lysine-coated 24-well plates 1 day before the assay at a density of 2 ϫ 10 5 cells per well. To assess the effect of PMA, the cultures were first incubated for 10 min in the absence (control) and presence of 1 M PMA in DMEM. Subsequently, the cells were washed once with pre-warmed EBSS. For the control group, the cells were further incubated with various concentrations of dopamine in a total volume of 0.4 ml at 37°C for 10 min in the presence of 3 M forskolin, 30 M Ro-20-1724 (phosphodiesterase inhibitor), 0.2 mM sodium metabisulfite (to prevent oxidation of dopamine), and 10 M propranolol (to block endogenous ␤-adrenergic receptors) in 20 mM HEPES-buffered DMEM. For the PMA-treated group, the cells were incubated with same composition of buffer as in the control group except for containing 1 M PMA. The supernatant was aspirated, and perchloric acid (3%, 200 l/well) was added. After incubating on ice for 30 min, 80 l of 15% KHCO 3 was added to the wells, and the plates were further incubated for 10 min. The plates were then centrifuged for 10 min at 1,300 ϫ g. Supernatant from each well was used for cAMP radioassay (Diagnostic Products Corp., Los Angeles, CA).
Data Analysis-All binding assays were routinely performed in triplicate and were repeated three to four times. Cyclic AMP experiments were performed in duplicate and were repeated three to four times. Estimation of the radioligand binding parameters, K D and B max , as well as the EC 50 values for dopamine inhibition of cAMP accumulation were calculated using the GraphPad Prizm curve-fitting program. The curves presented throughout this paper, representing the best fits to the data, were generated using this software program as well.

PKC-mediated Phosphorylation of D 2 DARs in HEK293T
Cells-As an initial approach to investigating the role of second messenger-activated protein kinases in D 2 DAR phosphorylation, we treated transiently transfected HEK293T cells with various activators and inhibitors of PKA and PKC (Fig. 1). The major phosphorylated protein in FLAG-tagged D 2 DAR-expressing cells runs as a broad band of 60 -85 kDa and is not present in immunoprecipitates of untransfected cells (data not shown). As seen in Fig. 1A, the D 2 DAR is phosphorylated under basal conditions, and treatment of the cells with PMA, a phorbol ester that directly activates PKC, increases the phosphate content of the D 2 DAR by ϳ3-fold (Fig. 1, A and B). Most interestingly, treatment of the cells with bisindolylmaleimide II (BIMII), an inhibitor of PKC, decreases the phosphorylation state of the D 2 DAR by about 50% (Fig. 1, A and B). In contrast, cellular treatment with agents that lead to PKA activation, including forskolin, which raises intracellular cAMP levels, and 8-(4-chlorophenylthio)adenosine 3Ј,5Ј cyclic AMP-cAMP, a membrane-permeable cAMP analog, have no effect on D 2 DAR phosphorylation ( Fig. 1, A and B). Similarly, treatment with H89, a PKA inhibitor, does not affect the phosphorylation state of the D 2 DAR (Fig. 1, A and B). These results suggest that PKC activation promotes D 2 DAR phosphorylation and that the basal receptor phosphorylation might be partially explained by constitutive PKC phosphorylation. The phosphorylation was specific for PKC, as the PMA effect was mimicked by PDBu but not by the biologically inactive phorbol ester 4␣PDD (Fig. 1C).
Agonist activation of the D 2 DAR has also been shown to promote its phosphorylation, an effect that is known to involve G-protein-coupled receptor kinases (18,19). Because in some cells D 2 DARs can stimulate phospholipase C-␤ to elevate intracellular calcium and diacylglycerol levels through activation of G-protein ␤␥ subunits (28,29), it is possible that agonistinduced D 2 DAR phosphorylation at least partly involves PKC. To investigate whether PKC-mediated phosphorylation is involved in the agonist-induced D 2 DAR phosphorylation, we incubated the cells with the PKC inhibitor BIMII before and during dopamine treatment. As shown in Fig. 2, dopamineinduced D 2 DAR phosphorylation was not affected by BIMII treatment, but PMA-stimulated D 2 DAR phosphorylation was totally abolished by the PKC inhibitor. Agonist activation using dopamine resulted in a doubling of the phosphorylation state of the D 2 DAR in both the absence or presence of BIMII (Fig. 2). Overall, this suggests that PKC is not involved in homologous phosphorylation of the D 2 DAR but may occur heterologously through the activation of G q -linked or other receptors that lead to PKC activation.
Because HEK293 cells do not abundantly express any known G q -linked receptors, we co-transfected the M1 muscarinic receptor, which couples to G q and leads to phospholipase C and PKC activation. Fig. 3, A and B, shows that in M1 receptor- transfected, but not in mock-transfected, cells, treatment with the muscarinic receptor agonist carbachol results in a 2-fold increase in D 2 DAR phosphorylation. These results show that activation of PKC through a G q -coupled receptor pathway can lead to phosphorylation of the D 2 DAR.
There are at least 12 isoforms of PKC that possess distinct differences in structure, substrate requirement, expression, and localization (30). It has been shown that HEK293 cells abundantly express the and isoforms of PKC and have moderate levels of the ␤I isoform (31). To elucidate which PKC isoform may be involved in D 2 DAR phosphorylation, we overexpressed several PKC isoforms with the D 2 DAR, and we examined their effects on receptor phosphorylation. Co-transfection of PKC␤I increased basal and PMA-stimulated D 2 DAR phosphorylation, whereas co-transfection of PKC␦, PKC, or PKC did not change D 2 DAR phosphorylation compared with mock (empty pcDNA vector) transfections (Fig. 3, C and D). In addition, we observed that cellular treatment with the PKC␤ isoform-specific inhibitor, LY333531 (32), abolished PMA-stimulated D 2 DAR phosphorylation (data not shown). These results suggest that the PKC␤ isoform is involved in D 2 DAR phosphorylation in the HEK293 cells.
Effect of PKC-mediated Phosphorylation on D 2 DAR Sequestration and Desensitization-As a first approach to investigating the effects of PKC-mediated phosphorylation on D 2 DAR function, we examined the expression of the receptor, both total cellular expression as well as just cell surface expression (Fig.  4). In order to assess the cell surface expression of the D 2 DAR, we used the radioligand, [ 3 H]sulpiride, and employed an intact cell binding assay. [ 3 H]Sulpiride is a hydrophilic antagonist ligand, which is membrane-impermeable, and restricted to binding only those receptors at the surface of intact cells (18,19,33). Fig. 4A shows that cellular treatment with PMA results in a 15-20% loss of D 2 DAR expression on the cell surface. In contrast, there is no effect of PMA treatment on total D 2 DAR expression as assessed using the hydrophobic antagonist [ 3 H]methylspiperone and membrane binding assays (Fig. 4B).  3. The effect of G q -coupled receptor activation or overexpressing various PKC isoforms on basal and PMA-induced D 2 DAR phosphorylation. Cells were transfected with pSF-D 2L along with pcDNA (empty vector) or mouse M1 muscarinic receptor cDNA or various PKC expression constructs. Whole-cell phosphorylation assays, autoradiography, and quantification were performed as described in Fig. 1 Many GPCRs have been shown to undergo sequestration or internalization from the cell surface via a ␤-arrestinand dynamin-dependent pathway generally as a result of GRK-mediated phosphorylation (4,34,35). GRK phosphorylation of many GPCRs results in their association with an arrestin-like protein that acts as a scaffold for the delivery of the receptors into clathrin-coated pits. Endocytosis of these pits requires the GTPase dynamin (36). This endocytic pathway, used by many GPCRs, can be disrupted by expressing dominant negative mutants of arrestins or dynamin. We thus evaluated the potential role of ␤-arrestin and dynamin in PMA-induced D 2 DAR internalization by co-expressing the dominant negative mutants, ␤-arrestin-(319 -418) (37) or dynaminK44A (38) with the D 2 DAR (Table II). As can be seen, the PMA-induced D 2 DAR sequestration is completely blocked through co-expression of either ␤-arrestin-(319 -418) or dynaminK44A. These results confirm the notion that PMA treatment promotes the sequestration of the D 2 DAR and further suggests that the internalization process is ␤-arrestin/dynamin-dependent and is mediated through a clathrin-coated pit pathway in the HEK293T cells.
We were next interested in testing for potential effects of PMA treatment on the functional coupling of the D 2 DAR. We thus examined D 2 DAR-mediated inhibition of forskolin-stimulated cAMP accumulation in the HEK293T cells. Fig. 5 shows that under basal conditions, dopamine is able to inhibit the forskolin-stimulated cAMP response by about 70%. Treatment of the cells with PMA did not affect the forskolin response per se (16.4 Ϯ 1.6 pmol/well for control; 14.3 Ϯ 2.4 pmol/well for PMA-treated) nor did it affect the maximum inhibition by dopamine (Fig. 5). In contrast, after PMA treatment, the EC 50 for dopamine inhibition of cAMP accumulation was shifted by about 3-fold to lower potency (Fig. 5). This appears to be because of a reduction in the potency of dopamine for eliciting this response rather than a decreased affinity for the receptor as competition radioligand binding assays did not reveal an alteration in the receptor binding affinity of dopamine (data not shown). These findings suggest that another consequence of PKC phosphorylation of the D 2 DAR is to attenuate functional G-protein coupling.
Identification of PKC Phosphorylation Sites within the D 2 DAR-We were next interested in attempting to identify the putative PKC phosphorylation sites in the D 2 DAR protein. PKC recognition and phosphorylation of proteins is known to require basic amino acid residues near the serine or threonine phosphoacceptor group(s) (39). We thus scanned the D 2 DAR sequence for all possible PKC recognition motifs, and the results are shown in Fig. 6. It should be noted that we discounted those serine and threonine residues within the 29-amino acid insertion sequence defining the D 2L DAR (see Fig. 6) as we did not observe any differences in basal or PMA-stimulated phosphorylation between the D 2S and D 2L isoforms (data not shown). To identify which residues were phosphorylated by PMA treatment, we mutated each putative consensus site, shown as black residues in Fig. 6, either individually or in combination. Comparable expression levels of all mutants were confirmed by radioligand binding assays, and identical results were obtained for immunoprecipitation and immunoblotting (data not shown). When the residues indicated in black, and with an asterisk (Fig. 6), were mutated, there was no change in PMA-stimulated receptor phosphorylation (data not shown), indicating that they do not serve as substrates for PKC. In contrast, when serines 228 and 229 (Fig. 6) were mutated to alanines, both basal and PMA-stimulated D 2 DAR phosphorylation was significantly diminished (Fig. 7, A and B). Individual mutation of either serine 228 or 229 resulted in a small reduc-   tion of receptor phosphorylation, whereas simultaneous mutation of both residues (mutant A) decreased the phosphorylation by about 50% compared with the wild-type receptor (Fig. 7, A  and B). These results suggest that serines 228 and 229 in the D 2 DAR are involved in the PKC-mediated phosphorylation but that additional residues are involved as well.
In order to identify the remaining PKC phosphorylation sites, we examined a cluster of residues, threonines 352 and 354 and serine 355, in a more distal segment of the third cytoplasmic loop of the receptor (Fig. 6). Fig. 7, C and D, shows that single mutations of each of these residues reduces the PKC-mediated phosphorylation of the receptor, although the greatest effect was observed with the single S355A mutant. These results suggest that there are two domains of PKC phosphorylation within the D 2 DAR. Domain I consists of serines 228 and 229 and is located in the proximal region of the third cytoplasmic loop (Fig. 6). Domain II consists of serine 355 and threonines 352 and 354 and is located in a more distal segment of the loop (Fig. 6).
To confirm that the domain I and II residues can fully account for the PKC-mediated phosphorylation, we analyzed various combinations of mutations, and the results are shown in Fig. 7, E and F. Each one of the mutant constructs shown in Fig. 7, E and F, 6. Diagram of the rat D 2L dopamine DAR sequence. Gray residues represent those absent in the D 2S isoform. Black residues represent putative PKC phosphorylation sites. Black residues with asterisks represent potential PKC phosphorylation sites and were mutated to alanine or valine residues, yet these mutations did not result in an alteration of PMA-induced phosphorylation. The numbered black residues correspond to the functionally identified PKC phosphorylation sites as determined by mutational analysis and as discussed in the text. The combination mutants, where more than one residue was mutated, are delineated at the bottom of the figure and are referred to as mutants A-F. phosphoacceptor amino acids within domains I and II (cf. Fig.  6). Clearly, the vast majority of the PMA-induced phosphorylation is reduced in mutant B, which involves serines 228, 229, and 355; however, when only threonines 352 and 354 are additionally mutated is there a complete loss of the PMA effect (Fig. 7, E and F). These results confirm the notion that PKC phosphorylation of the D 2 DAR takes place on two internal domains primarily involving serine residues 228, 229, and 355 but with an additional contribution of threonine residues 352 and 354.
The Role of Specific PKC Phosphorylation Sites in PMA Regulation of the D 2 Receptor-Because PKC can phosphorylate and regulate many cellular signaling molecules involved in D 2 receptor signaling such as G␣ i (40,41), GRK2 (42), or adenylyl cyclase 2 (43), we wanted to correlate the specific PKC phosphorylation sites on the receptor with the PMA-induced regulatory effects. We initially examined various phosphorylation-defective mutant receptors on PMA-induced receptor sequestration (Fig. 8). There was no difference in the basal cell surface receptor expression levels between wild-type and all the mutant receptors (data not shown). Mutation of serines 228 and 229 to alanines (MutA) diminished PMA-induced D 2 DAR sequestration compared with that of wild-type receptor. In contrast, mutation of serine 355 appeared to have no effect on PMA-induced receptor sequestration as evidenced by the single mutant receptor (S355A) or when mutated in combination with serines 228/229 (MutB). Most interestingly, a mutant with the three domain II substitutions (MutC), which includes S355A, also showed diminished receptor sequestration. This result would imply a role for threonine residues 352 and 354 in the sequestration response. When all five domain I and II residues are mutated (MutF), the PMA-induced sequestration is essentially abolished (Fig. 8). Taken together, these results suggest that both of the PKC phosphorylation domains on the D 2 DAR are involved in regulating its sequestration from the cell surface.
We next evaluated PKC phosphorylation site-defective D 2 DARs for their ability to undergo PMA-induced desensitization (Fig. 9). When all potential PKC phosphorylation sites from domains I and II are simultaneously mutated (MutF), the PMA-induced EC 50 shift for dopamine is abolished. The loss of this regulatory response appears to be due to mutation of a single residue, serine 355, as the single S355A mutant shows the same complete loss of response as MutF (Fig. 9). These results indicate that, in contrast to the PMA-induced sequestration response, only serine 355 in domain II is involved in the PMA-induced desensitization. DISCUSSION In our present study, we have identified and characterized two domains of PKC phosphorylation sites within the D 2 DAR and correlated their phosphorylation with receptor desensitization and internalization. Previously, little information has been available concerning the role of regulatory phosphorylation of the D 2 DAR or specific kinases involved. GRKs 2 and 3 FIG. 7. Identification of PKC phosphorylation sites in the D 2 DAR. 32 PO 4 -Labeled cells were incubated in the absence or presence of 1 M PMA for 15 min, subjected to immunoprecipitation as described under "Experimental Procedures," and resolved by 4 -20% SDS-PAGE. Autoradiograms from single experiments are shown, which were quantified by scanning the autoradiographs followed by analysis with Lab-Works TM software (UVP Inc.). A, whole-cell phosphorylation assays were performed by using the indicated WT or single (S228A or S229A) or double (Mutant A (MutA) mutant D 2 DARs. B, data are presented as a percentage above basal phosphorylation of the WT D 2L receptor and expressed as the mean Ϯ S.E. from at least four independent experiments. C, whole-cell phosphorylation assays were performed using the WT or single (S355A, T352V, or T354V) mutant D 2 DARs. D, data are presented as a percentage above basal phosphorylation of the WT D 2L DAR and expressed as the mean Ϯ S.E. from at least four independent experiments. E, whole-cell phosphorylation assays were performed indicated WT or mutant D 2 DARs as defined in Fig. 6. Note that each of these mutant constructs contains the double serine mutation from domain I along with selected mutated residues from domain II. See  Fig. 4 and Table I have been shown to phosphorylate the D 2 DAR leading to enhanced agonist-stimulated receptor internalization (18,19). GRKs 5 and 6 have been suggested to regulate D 2 DAR sequestration or desensitization, although direct phosphorylation of the receptor by these kinases has not yet been demonstrated (18,44). Our investigation is now the first to demonstrate PKC-mediated phosphorylation of the D 2 DAR and the first to delineate specific phosphorylation sites within the D 2 DAR that regulate functional coupling and intracellular trafficking. These results may explain previous reports of phorbol ester effects on D 2 DAR functioning (20 -24, 45) and also provide a mechanism for how the D 2 DAR may be regulated by heterologous desensitization through activation of G q -coupled GPCRs. In fact, our present results show that agonist activation of the M1 muscarinic receptor can lead to D 2 DAR phosphorylation. Furthermore, recent studies using endogenous tissues have suggested that the G q -coupled cholecystokinin CCK 2 (46) and the neurotensin NT1 (47) receptors can negatively modulate D 2 DAR function, possibly through a PKC-mediated mechanism.
It was interesting to find that two domains of PKC phosphorylation sites exist within the D 2 DAR, both of which control receptor sequestration, whereas only one (domain II) is associated with receptor desensitization. Furthermore, within domain II only serine 355 seemed to be associated with functional uncoupling of the receptor. How phosphorylation of this residue leads to decreased G-protein coupling is unclear at this time. However, this residue is in close proximity to the distal end of the 3rd cytoplasmic loop near the sixth transmembrane domain. We (48) and others (49) have shown that both the proximal and distal regions of the 3rd cytoplasmic loop of the D 2 DAR, near the transmembrane segments, are involved in Gprotein coupling. Thus, one hypothesis is that phosphorylation of serine 355 disrupts G-protein coupling of this distal segment of the loop either through an electrostatic or conformational mechanism leading to impaired G-protein coupling and decreased potency for inhibition of adenylyl cyclase activity. This would be similar to the proposed mechanism for PKA-mediated phosphorylation and desensitization of the ␤ 2 -adrenergic receptor (50).
Similarly, the mechanism by which PKC phosphorylation of residues in domains I and II leads to receptor sequestration/ internalization remains to be determined. Our results indicate that the PKC-promoted receptor internalization occurs through a ␤-arrestinand dynamin-dependent pathway suggesting the involvement of clathrin-coated pits. Previously, the D 2 DAR has been shown to undergo agonist-induced internalization via a dynamin-dependent mechanism (19, 51, 52; however, see 53). One possibility is that phosphorylation of the 3rd cytoplasmic loop by PKC promotes ␤-arrestin association in a fashion similar to, but independent from, that proposed for GRK phosphorylation (19). In this case, multisite phosphorylation of the 3rd cytoplasmic loop by PKC would enable its direct association with ␤-arrestin leading to receptor internalization. This would represent a novel mechanism for PKC-mediated regulation of GPCRs, which is more usually associated with functional desensitization rather than receptor internalization.
Alternatively, another consideration is that the D 2 DAR is proposed to exhibit constitutive activity (54,55) that may be associated with constitutive GRK phosphorylation and internalization as suggested for constitutively active GPCRs (56,57). Indeed, the D 2 DAR has been shown previously to exhibit constitutive agonist-independent internalization and recycling (53). Thus, another possibility is that PKC phosphorylation facilitates or enhances the constitutive GRK-mediated pathway leading to increased basal internalization. As GRKs prefer to phosphorylate serine or threonine residues in close proximity to negatively charged residues, one possibility is that prior phosphorylation by PKC enhances the affinity of GRKs for neighboring serines or threonines, the phosphorylation of which leads to ␤-arrestin association. Experiments designed to differentiate these various possibilities are currently in progress.
It was of interest that we did not observe any evidence for D 2 receptor phosphorylation by PKA. This was somewhat surprising given that there are multiple consensus recognition sequences for PKA within the D 2 DAR protein, and an earlier report (22) using brain membranes suggested that PKA could negatively modulate D 2 DAR function. One possible explanation for this observation would be if HEK293 cells did not express high levels of PKA; however, these cells have been used previously to study PKA-mediated phosphorylation of ␤ 2 -adrenergic receptors (50). Another possibility is that HEK293 cells lack a necessary adaptor or auxiliary protein that might specifically link PKA to the D 2 DAR. This might be evaluated by using other cell lines to examine D 2 DAR phosphorylation in response to PKA activation. Alternatively, the D 2 DAR may not be a good substrate for PKA and might not be endogenously regulated by this protein kinase.
Our present results also suggest that there is specificity associated with PKC phosphorylation of the D 2 DAR. Previously, HEK293 cells were reported as expressing the , , and ␤I isoforms of PKC (31). Consequently, we overexpressed each of these isoforms, as well as PKC␦, and found that only the ␤I isoform leads to increased D 2 DAR phosphorylation. Co-expression of this isoform also leads to increased receptor internalization in response to PMA. Additionally, a PKC␤-specific inhibitor was found to block PMA-induced D 2 DAR phosphorylation. Although it is possible that other, untested, isoforms of PKC might also be capable of phosphorylating the D 2 DAR, it is clear that the ␤I isoform can specifically mediate this response. PKC␤I is expressed ubiquitously in multiple tissues and is found at high levels in the brain (58). It will be interesting in future experiments to determine the exact cellular colocalization of the D 2 DAR and ␤I isoform of PKC. In cells expressing high levels of PKC␤I, the D 2 DAR may be particularly susceptible to this form of heterologous regulation.
In summary, we have found that PKC can mediate phosphorylation of the D 2 DAR producing novel functional effects. This regulatory event appears to be specific for the ␤I isoform of PKC, although additional isoforms remain to be tested. Two phosphorylation domains, each containing a cluster of serine or threonine residues, were identified within the 3rd cytoplasmic domain of the receptor. Both of these domains regulate internalization of the receptor, whereas only one is involved in receptor desensitization. These results provide a mechanism by which the D 2 DAR can be regulated in a heterologous fashion through receptors that activate PKC signaling pathways.