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The D₁ Dopamine Receptor Is Constitutively Phosphorylated by G Protein-Coupled Receptor Kinase 4

Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland (M.L.R., P.S.M., D.M.C., D.R.S.); and Department of Pediatrics, Georgetown University Medical Center, Washington DC (Z.W., P.A.J.)

Received October 17, 2005; accepted December 7, 2005

	Abstract

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G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D₁ receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the isoform of GRK4; the , , and isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4 is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D₁ receptor by GRK4 results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number—all of which are abolished in a D₁ receptor mutant containing T428V and S431A. The increase in internalized D₁ receptors induced by GRK4 phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D₁ receptor by GRK4 does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4. These observations shift the attenuation of D₁ receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization.

Upon agonist activation, GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response under continued agonist stimulation (Ferguson et al., 1996; Gainetdinov et al., 2004). Desensitization involves phosphorylation of the receptor by GRKs and/or second messenger-activated kinases (cAMP-dependent protein kinase or protein kinase C). Homologous desensitization of GPCRs involves only activated receptors and is primarily mediated by GRKs. GRKs are serine/threonine-directed protein kinases composed of seven isoforms divided into three families (Penela et al., 2003). GRK1 and GRK7 compose the rhodopsin kinase/visual family, are expressed exclusively in retina, and participate in desensitization of opsins in rods and cones (Somers and Klein, 1984; Hisatomi et al., 1998; Weiss et al., 1998). GRK2 (ARK1) and GRK3 (ARK2) were originally identified as regulating the -adrenergic receptor and compose the ARK family. The GRK4 family consists of GRK4, GRK5, and GRK6. Four RNA splice variants have been identified for GRK4: , , , and (Premont et al., 1996). GRK4 represents the largest member; , , and each represent different truncations of the sequence. GRK-mediated phosphorylation has been shown to decrease receptor/G protein interactions and to initiate arrestin binding. Arrestin association further decreases G protein coupling and promotes endocytosis of the receptor. Once internalized, GPCRs can engage additional signaling pathways, be sorted for recycling to the plasma membrane, or targeted for degradation (Ferguson et al., 1996; Penela et al., 2003; Gainetdinov et al., 2004).

As with most GPCRs, the D₁ dopamine receptor is known to undergo desensitization and internalization, and these events have been studied in a variety of systems. A preponderance of evidence indicates that desensitization/internalization of the D₁ receptor is regulated by its phosphorylation state; this, in turn, is mediated by both second messenger-activated kinases as well as by GRKs. Phosphorylation of the D₁ receptor by the cAMP-dependent protein kinase seems to occur on a single threonine residue, Thr268, within the 3rd cytoplasmic loop, and this modification regulates the rate of agonist-induced desensitization (Jiang and Sibley, 1999) and/or the intracellular trafficking of the receptor (Mason et al., 2002). In contrast, evidence concerning the nature, number, and location of GRK phosphorylation sites within the D₁ receptor is contradictory. Jackson et al. (2002) have suggested that the D₁ receptor is phosphorylated by GRKs on multiple residues within its carboxyl terminus. Likewise, Lamey et al. (2002) have provided evidence that agonist-induced phosphorylation of the human D₁ receptor is restricted to the carboxyl terminus; however, they report that only a single residue, Thr360, is involved. In contrast, Tiberi et al. (1996) observed that GRK-mediated phosphorylation of the D₁ receptor takes place exclusively on serine residues. We have recently suggested that agonist-induced, GRK-mediated phosphorylation of the D₁ receptor occurs on residues within both the carboxyl terminus and the third cytoplasmic loop and that the phosphorylation of these domains may take place in an ordered fashion (Kim et al., 2004). It is conceivable that these different results might be due to the expression of different complements of GRK isoforms in the various cell systems under study.

The D₁ dopamine receptor seems to be a substrate for at least GRK2-5 (Tiberi et al., 1996; Felder et al., 2002). To investigate whether these different GRKs differentially phosphorylate the D₁ receptor, we coexpressed each of the nonvisual GRKs with the D₁ receptor and examined its phosphorylation state in the absence or presence of agonist. We were surprised to find that GRK4 coexpression resulted in phosphorylation of the receptor in the basal state to a very high level that was only marginally increased upon addition of agonist. This constitutive phosphorylation of the D₁ receptor was observed only with the isoform of GRK4. This agonist-independent phosphorylation of the D₁ receptor by GRK4 was found to result in a decrease in agonist-induced cAMP accumulation, an increase in basal receptor internalization, and a decrease in total receptor number. Our present results with GRK4 and the D₁ receptor contrast with the existing dogma that GRKs phosphorylate only agonist-occupied/activated GPCRs and provide a novel mechanism whereby GRKs can regulate GPCR function.

	Materials and Methods

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Materials. HEK293-tsa201 (HEK293T) cells (Heinzel et al., 1988) were a gift from Dr. Vanitha Ramakrishnan. Rat D₁-GFP was a gift from Dr. Qun-Yong Zhou (Bermak et al., 2002). GRK4 isotypes were obtained from Dr. Richard Premont. GRK4 single-nucleotide polymorphisms (SNPs) were as described previously (Felder et al., 2002). GRK2 and GRK3 clones as well as the dominant-negative -arrestin minigene construct (-arrestin 319-418) were obtained from Dr. Jeffrey L. Benovic. [³H]SCH-23390 (86.00 Ci/mmol) and [³²P]orthophosphate (carrier-free) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). [³H]cAMP (31.4 Ci/mmol) was purchased from Diagnostic Products Corporation (Los Angeles, CA). Dopamine, Ro-20-1724, sodium metabisulfite, and anti-FLAG M2 affinity gel were purchased from Sigma (St. Louis, MO). (+)-Butaclamol was purchased from Research Biochemicals Inc. (Natick, MA). Cyclic AMP assay kits were obtained from Diagnostic Products Corp. (Los Angeles, CA). Calcium phosphate transfection kits were obtained from Clontech (Mountain View, CA). MiniComplete protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN). QuikChange and QuikChange Multi site-directed mutagenesis kits were purchased from Stratagene (La Jolla, CA), and mutagenesis primers were synthesized by MWG Biotech Inc. (High Point, NC). Cell culture media and reagents, NuPage gels, buffers, and Invitrolon 0.45-µm PVDF membranes were purchased from Invitrogen (Carlsbad, CA). Anti-GRK4 (H-70) rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), peroxidase-conjugated goat anti-rabbit IgG secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and SuperSignal West Dura extended duration substrate antibodies kit was purchased from Pierce (Rockford, IL).

Cell Culture and Transfection. HEK293T cells were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10 µg/ml gentamycin. Cells were grown at 37°C in 5% CO₂ and 90% humidity. An amino terminal FLAG epitope-tagged construct for the rat D₁ receptor (Monsma et al., 1990) was created from pSF₂, an expression construct containing a FLAG-tagged ₂-adrenergic receptor (Guan et al., 1992) to create the wild-type pSFD₁ as reported previously (Gardner et al., 2001). This construct, and mutants thereof, were used for all experiments except for the confocal fluorescence microscopy experiments in which the D₁-GFP construct was used. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kits. For phosphorylation mutants, serine residues were mutated to alanine residues and threonine residues were mutated to valine residues. All mutant FLAG-D₁ constructs were verified by DNA sequence analysis before use. HEK293T cells were transfected using the calcium phosphate precipitation method (Clontech). After 24 h, the transfected cells were divided and reseeded for subsequent experiments on the following day.

Whole-Cell Phosphorylation Assay. These assays were performed as described previously (Gardner et al., 2001). In brief, 1 day before the experiment, transfected HEK293T cells were seeded at 1.5 x 10⁶ cells per well of a six-well poly-D-lysine-coated plate, and 2 x 10⁶ cells of the same transfection were seeded in a 100-mm culture dish and cultured overnight. Cells in the six-well plates were washed with EBSS and incubated for 1 h in phosphate-free DMEM. Media was then removed and replaced with 1 ml of fresh phosphate-free DMEM containing 106 µCi of [³²P]H₃PO₄ and returned to the incubator for 45 min. This procedure has been shown previously to result in stoichiometric phosphorylation of the D₁ receptor (Gardner et al., 2001). Cells were then challenged with 10 µM dopamine in the presence of 0.2 mM sodium metabisulfite for 15 min then placed on ice. Cells were 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, 50 mM NaF, 40 mM sodium pyrophosphate, and 150 mM NaCl) supplemented with Mini-Complete protease inhibitor cocktail. The samples were cleared by centrifugation and the protein concentration was determined using the BCA protein assay kit from Pierce. The specific activity of D₁ receptor expression in each transfection group was determined by radioligand binding assays performed using cells in the 100-mm culture dishes described above. After quantifying the receptors in each transfection group, equal amounts of receptor protein were transferred to fresh tubes containing 50 µl of equilibrated anti-FLAG M2-affinity gel and incubated overnight with mixing at 4°C. The samples were washed three times with the following solutions in the order of 0.5 M NaCl solubilization buffer, 0.15 M NaCl solubilization buffer, and Tris-EDTA, pH 7.4, at 4°C. Proteins were eluted from the affinity gel by addition of 35 µl of 2x lithium dodecyl sulfate sample buffer containing reducing agent (Invitrogen) and incubated at 37°C for 1 h. Proteins were resolved on 4 to 12% NuPage Bis-Tris gradient gels run in MOPS SDS running buffer (Invitrogen), dried, and subjected to autoradiography.

Western Blotting. HEK293T cells were cultured, transfected, and seeded into six-well plates as described for the whole-cell phosphorylation assays above. Cells were washed twice with ice-cold EBSS and solubilized for 1 h at 4°C in 0.5 ml of solubilization buffer (50 mM HEPES, 1 mM EDTA, 10% glycerol, 1% Triton X-100, pH 7.4, 50 mM NaF, 40 mM sodium pyrophosphate, and 150 mM NaCl) supplemented with MiniComplete protease inhibitor cocktail. The samples were cleared by centrifugation and the protein concentration was determined using the BCA protein assay kit from Pierce. A concentrated stock of 4x lithium dodecyl sulfate sample buffer containing reducing agent (Invitrogen) was prepared and added to the cell lysate volume that corresponded to 45 µg of total protein per cell lysate and incubated at 37°C for 1 h. Proteins were resolved on 4 to 12% NuPage Bis-Tris gradient gels run in MOPS SDS running buffer (Invitrogen), transferred to Invitrolon 0.45-µm PVDF membranes in NuPage transfer buffer containing methanol, according to the manufacturer's instructions, and blocked overnight at 4°C in a 1% BSA solution made in TBST. For identification of GRK4 proteins within the cell lysates, the PVDF membranes were incubated in a primary antibody solution of GRK4 (H-70) rabbit polyclonal antibody diluted 1:500 in 1% bovine serum albumin/TBST solution for 1 h at room temperature. The membranes were washed with TBST then incubated in a secondary antibody solution of peroxidase-conjugated goat anti-rabbit diluted 1:25,000 in 1% bovine serum albumin/TBST solution for 1 h at room temperature and washed with TBST. Western blots were developed using the Super Signal West Dura extended duration substrate antibody kit from Pierce (Rockford, IL).

Radioligand Binding Assays. HEK293T cells were harvested by incubation with 5 mM EDTA in EBSS lacking CaCl₂ and MgSO₄ and collected by centrifugation at 300g for 10 min. The cells were resuspended in lysis buffer (5 mM Tris, pH 7.4, and 5 mM MgCl₂) at 4°C and were disrupted using a Dounce homogenizer followed by centrifugation at 34,000g for 15 min. The resulting membrane pellet was resuspended in binding buffer (50 mM Tris, pH 7.4), and 100 µl of the membrane suspension was added to assay tubes containing [³H]SCH-23390 in a final volume of 1 ml. A portion of the membrane suspension was quantitated using the BCA protein assay (Pierce). (+)-Butaclamol was added at a final concentration of 3 µM 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 GC/F filters pretreated with 0.6% polyethylenimine. Radioactivity bound to filters was quantitated by liquid scintillation spectroscopy at a counting efficiency of 47 to 60%.

Determination of cAMP Production. Transfected HEK293T cells were seeded into 24-well poly-D-lysine-coated plates (100,000 cells/well) and cultured for 1 day before experimentation. To assess desensitization, cells were pretreated with 10 µM dopamine with 0.2 mM sodium metabisulfite for 1 h and then washed three times with 200 µl of EBSS per well. Various concentrations of dopamine were added to each well in a final volume of 250 µl containing 50 µM Ro-20-1724 and 0.2 mM sodium metabisulfite and placed at 37°C for 20 min. The reaction was terminated by adding 200 µl of 3% per-chloric acid and incubated on ice for 30 min before adding 80 µl of 15% KHCO₃. The plates remained on ice for an additional 20 min and were centrifuged at 1300g for 10 min. Supernatant (50 µl) from each well was transferred to a 1.2-ml tube containing 150 µl of Tris-EDTA buffer, 50 µl of cAMP binding protein, and 50 µl of [³H]cAMP and incubated overnight at 4°C. Charcoal-dextran mix (1%; 250 µl) was added to each tube and gently vortexed. Tubes were incubated at 4°C for 10 min then centrifuged at 1300g for 20 min. Radioactivity in the supernatant from each tube was quantified by liquid scintillation spectroscopy at a counting efficiency of 47 to 60%. cAMP concentrations were calculated using a standard curve according to the protocol of the assay kit.

Confocal Microscopy. HEK293T cells (3 x 10⁵) were seeded in 100-mm culture dishes. The next day, the cells were transfected with 300 ng of D₁-GFP in pEGFP-N1 vector with or without 4 to 5 µg of GRK4 and 3 µg of dominant-negative -arrestin (319-418) then cultured for an additional 24 h before reseeding the transfections at 100,000 cells per poly-D-lysine-coated, glass-bottomed, 35-mm culture dish. Before stimulation, the media was replaced with Opti-MEM I reduced serum medium. Confocal microscopy was performed on a Zeiss laser-scanning confocal microscope (LSM-510; Zeiss, Welwyn Garden City, UK). Images were collected sequentially every 30 s for a total of 15 min after agonist stimulation with 20 µM dopamine using a single line excitation (488 nm).

Data Analysis. All phosphorylation assays were performed at least three times. Figures depict representative autoradiography obtained for each experimental condition. Relative intensities of the phosphorylated bands were determined by scanning the autoradiographs and analyzing the bands using LabWorks 4.0 (UVP, Inc., Upland, CA). Binding assays and cAMP experiments were performed three to four times. Radioligand binding parameters K_D and B_max,as well as EC₅₀ values for dopamine-stimulated cAMP production, were calculated using the curve-fitting program Prism (version 3.02; GraphPad, San Diego, CA).

	Results

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GRK-Mediated Phosphorylation of the D₁ DAR in HEK293T Cells. To study GRK-mediated phosphorylation of the D₁ DAR, we transiently cotransfected HEK293T cells with the FLAG-tagged D₁ DAR and G protein-coupled receptor kinases GRK2, GRK3, and GRK4. Empty expression vector was cotransfected as a control for the GRK cotransfection experiments. The transfected cells were then subjected to whole-cell phosphorylation assays to monitor phosphate incorporation into the D₁ DAR under basal conditions and in the presence of agonist. The D₁ DAR was phosphorylated under basal conditions, and this phosphorylation was increased upon cotransfection with various GRK subtypes (Fig. 1, A and B). Upon exposure to agonist (10 µM dopamine for 15 min), control cells expressing D₁ DAR alone displayed a 4-fold increase in receptor phosphate content, whereas cells coexpressing either GRK2 or GRK3 displayed a 7- to 12-fold increase in receptor phosphorylation. We were surprised to find that cells coexpressing D₁ DAR and GRK4 displayed a 4-fold increase in receptor phosphate content in the basal state compared with control, and this amount of phosphorylation was increased only marginally upon exposure to agonist. HEK293T cells cotransfected with the D₁ DAR and either GRK5 or GRK6 did not display phosphorylation patterns (basal or agonist-stimulated) that differed significantly from control cells (data not shown). These data show that coexpression of GRK4 with D₁ DAR results in an increase in basal receptor phosphorylation that is comparable with control cells exposed to agonist.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Effect of coexpressing individual GRK isoforms on D1 receptor phosphorylation. HEK293T cells were transfected with wild-type (WT) FLAG-tagged D1 receptor and empty vector (V) or GRK2 (+2), GRK3 (+3) or GRK4 (+4). Transfected cells were metabolically labeled with [32P]H3PO4 for 45 min before stimulation with 10 µM dopamine (DA) for 15 min. Samples were then subjected to immunoprecipitation and resolved on 4 to 12% NuPage BT gels before autoradiography as described under Materials and Methods. Receptors were quantified in each transfection before gel electrophoresis to ensure equal amounts of D1 receptor in each lane. A, autoradiogram of D1 receptor immunoprecipitates from whole cell phosphorylation assays. D1 DAR (1 pmol) was loaded in each lane. A representative experiment (performed three to five times) is shown. B, the receptor phosphorylation obtained in A was quantified by scanning the autoradiographs followed by analysis with LabWorks software (UVP Inc.). Data are presented as the -fold increase in phosphorylation above the WT basal condition and expressed as mean ± S.E.M. values from four independent experiments.

GRK4 Subtype-Specific Phosphorylation of the D₁ DAR. The GRK4 subfamily displays a number of alternative RNA splice variants (Ambrose et al., 1992; Premont et al., 1996, 1999). GRK4 is the largest member of the GRK4 subgroup with alternative splicing resulting in the smaller , , and splice variants (Fig. 2). To determine whether these shorter GRK4 splice variants could also mediate agonist-independent phosphorylation of the D₁ DAR, HEK293T cells were cotransfected with the D₁ DAR and each GRK4 splice variant and then subjected to whole-cell phosphorylation assays. Only the GRK4 splice variant displayed agonist-independent phosphorylation of the D₁ DAR whereas the GRK4, -, and - subtypes had no effect (Fig. 3A). We also examined three SNPs of GRK4 that have been reported to regulate desensitization of the D₁ DAR (Felder et al., 2002). We performed similar whole-cell phosphorylation experiments as shown in Fig. 3A with transfected HEK293T cells expressing D₁ DAR and three GRK4 SNPs (R65L, A142V, and A486V). None of the GRK4 SNPs mediated phosphorylation of the D₁ DAR in a manner significantly different from that of control cells (Fig. 3A). To be sure that the inability of the shorter GRK4 subtypes to induce agonist-independent phosphorylation of the D₁ DAR was not due to less efficient expression of these constructs, Western blotting experiments were performed to monitor the level of GRK4 isoform expression. HEK293T cells were cotransfected with the four GRK4 splice variants as performed in Fig. 3A. These cells were subjected to Western blotting analysis using a primary antibody that detects all GRK4 splice variants. Cells transfected with GRK4, -, -, or - all displayed equally robust levels of GRK4 protein expression (Fig. 3B). To determine whether increasing GRK4, -, -, or GRK4 SNP expression would result in an increase in basal D₁ DAR phosphorylation, HEK293T cells were cotransfected with 2- and 4-fold the amount of DNA used initially to boost the expression of the GRK4 variants. Increasing the amount of GRK4, -, and -, or GRK4 R65L, A142V, and A486V SNPs did not result in constitutive phosphorylation of the D₁ DAR (Fig. 3, C and D; data not shown for SNPs A142V and A486V). These data indicate that only the GRK4 subtype mediates constitutive phosphorylation of the D₁ DAR in the absence of agonist stimulation in HEK293T cells.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Diagram of the human GRK4 splice variants. The diagram represents the amino acid sequence of the four GRK4 isoforms that result from alternative splicing of the GRK4 mRNA originally identified by (Ambrose et al., 1992; Premont et al., 1999). Boxes (clear/hatched) and lines (solid/dashed) represent identical amino acid sequences.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Phosphorylation of the D1 receptor coexpressed with individual GRK4 isoforms and SNPs. HEK293T cells were transfected with WT FLAG-tagged D1 receptor and either empty V or various GRK4 constructs. Transfected cells were metabolically labeled with [32P]H3PO4 for 45 min before stimulation with 10 µM dopamine (DA) for 15 min. A, autoradiograms of D1 receptor immunoprecipitates from whole-cell phosphorylation assays. HEK293T cells were transfected with WT D1 receptor and empty V or human GRK4 splice variants (, , , and ) or human GRK4 SNPs (R65L, A142V, and A486V). D1 DAR (1 pmol) was loaded in each lane. A single experiment representative of four is shown. B, Western blot of cells transfected as in A with D1 receptor and empty V or human GRK4 splice variants (, , , and ). Cell lysate (45 µg) was loaded in each lane and visualized as described under Materials and Methods. The antibody used is directed to an epitope common to all isoforms. C, cells were treated as in A except that GRK4 and GRK4 isoforms were transfected using 2- or 4-fold the amount of DNA compared with empty vector or the GRK4 isoform. D1 DAR (1 pmol) was loaded in each lane. A single experiment representative of three is shown. D, cells were treated as in C except that increasing amounts of GRK4 or the GRK4 R65L SNP were employed. D1 DAR (1 pmol) was loaded in each lane. A single experiment representative of three is shown.

Identification of the GRK4 Phosphorylation Sites within the D₁ DAR. Two regions of the D₁ DAR have been implicated in mediating GRK phosphorylation—the third intracellular loop and the long carboxyl terminus (Jackson et al., 2002; Lamey et al., 2002; Kim et al., 2004). To determine the sites of GRK4-mediated phosphorylation within the D₁ DAR, various receptor mutants were constructed using site-directed mutagenesis and coexpressed with GRK4 in HEK293T cells followed by whole-cell phosphorylation assays. Figure 4 depicts all of the receptor mutants that were used in this study.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Diagram of the rat D1 receptor sequence. The wild-type receptor sequence is shown along with the various mutant receptor constructs used in this study. Four carboxyl terminal truncation mutants were generated by inserting a stop codon after amino acids 347, 369, 394, or 404 (T347, T369, T394, and T404). Shaded circles indicate serine or threonine residues mutated to alanine or valine, respectively, in various combinations. Third intracellular loop mutants include the individual serine point mutations (S2, S3, and S4), the S234 mutant that includes all three point mutants S2, S3, and S4. In the 3rd TOT mutant, all of the serines and threonines in the third intracellular loop were simultaneously mutated. Individual point mutations in the distal carboxyl tail region include S417A, S421A, T428V, S431A, T439V, S441A, S445A, and T446V, whereas the mutT404 mutant contains all of these point mutations with in a single construct. D1 DAR cluster mutant 1516 contains T428V and S431A point mutations, and cluster mutant 181920 contains S441A, S445A, and T446V point mutations as indicated.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Effect of GRK4 on phosphorylation of wild-type and mutant D1 receptors. HEK293T cells were transfected with either WT or mutant D1 receptors and either empty V or GRK4 (+4). Transfected cells were metabolically labeled with [32P]H3PO4 for 45 min before stimulation with 10 µM dopamine (DA) for 15 min. In each case, WT D1 receptor cotransfected with either empty V or GRK4 are included as controls. A, autoradiogram of D1 receptor immunoprecipitates from whole cell phosphorylation assays using the carboxyl terminal truncation mutants (see Fig. 4 for nomenclature). D1 DAR (0.5 pmol) was loaded in each lane. A single experiment representative of three is shown. B, autoradiogram of D1 receptor immunoprecipitates from whole-cell phosphorylation assays using the third intracellular loop mutants (see Fig. 4 for nomenclature). D1 DAR (1.5 pmol) was loaded in each lane. A single experiment representative of three is shown. C, autoradiogram of D1 receptor immunoprecipitates from whole cell phosphorylation assays using mutT404 and T404. D1 DAR (2 pmol) was loaded in each lane. A single experiment representative of three is shown.

To further identify the specific D₁ DAR residues phosphorylated by GRK4 in the absence of agonist, each of the serine and threonine residues in the region between amino acid 404 and the carboxyl terminus of the receptor was individually mutated as depicted in Fig. 4. Preliminary experiments revealed that no single point mutation resulted in complete abolishment of GRK4-mediated constitutive phosphorylation of the D₁ DAR; however, some point mutants seemed to be more effective than others. In particular, point mutants S417A, S421A, and T439V showed little or no reduction in constitutive phosphorylation by GRK4; mutants T428V, S431A, S441A, S445A, and T446V, however, each displayed some degree of reduced constitutive phosphorylation (data not shown). To further investigate this effect, two D₁ DAR cluster mutants were created based on the location of the single point mutants that showed some reduction in GRK4-mediated constitutive phosphorylation. D₁ DAR mutant "1516" contains the T428V and S431A mutations, and D₁ DAR mutant "181920" contains the S441A, S445A, and T446V mutations (Fig. 4). Each D₁ DAR cluster mutant was coexpressed with GRK4 and subjected to whole-cell phosphorylation assays in the absence and presence of dopamine (Fig. 6). As shown previously, dopamine-stimulation increases the phosphate content of the WT D₁ DAR by approximately 3-fold in the absence of GRK4, whereas coexpression with GRK4 increases the basal phosphorylation and nearly eliminates the effect of dopamine stimulation. As can be seen, the two cluster mutants behave more similarly to the wild-type D₁ DAR (Fig. 6) and the mutant constructs T404 and mutT404 (Fig. 5C). This effect is particularly striking for the 1516 cluster mutant in which Thr428 and Ser431 are simultaneously mutated (Fig. 6B). These results suggest that five serine and threonine residues in the distal carboxyl terminus of the D₁ DAR contribute to the constitutive phosphorylation by GRK4 with a primary involvement of Thr428 and Ser431.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Effect of GRK4 on phosphorylation of wild-type and D1 DAR cluster mutants. HEK293T cells were transfected with either WT or cluster mutant (1516 or 181920) D1 receptor constructs (see Fig. 4) and either empty V or GRK4 (+4). Transfected cells were metabolically labeled with [32P]H3PO4 for 45 min before stimulation with 10 µM dopamine for 15 min. A, autoradiogram of D1 receptor immunoprecipitates from whole cell phosphorylation assays. D1 DAR (1.5 pmol) was loaded in each lane. A representative experiment (performed four times) is shown. B, the receptor phosphorylation (intensity) obtained for each transfection was quantified by scanning the autoradiograms followed by analysis with LabWorks software (UVP Inc.). The -fold increase in phosphorylation in response to dopamine stimulation was calculated by dividing the intensity of the phosphorylation in the dopamine-stimulated groups by the phosphorylation seen in nontreated groups (basal). An experimental result showing no increase of phosphorylation over basal would yield a value of 1.0. The data are expressed as mean ± S.E.M. values from four independent experiments.

Effect of GRK4-Mediated Phosphorylation of D₁ DAR on cAMP Accumulation and Desensitization. Having observed the unique nature of GRK4-mediated phosphorylation of the D₁ DAR, we next studied its effects on dopamine-stimulated cAMP production. Coexpression of the wild-type D₁ DAR with GRK4 resulted in a significant decrease in dopamine-stimulated cAMP accumulation (Fig. 7A). This reduction in D₁ DAR-mediated cAMP accumulation by GRK4 was largely absent in similar experiments using the D₁ DAR truncation mutant T404 and the corresponding mutT404 mutant (Fig. 7, B and C), neither of which exhibited constitutive GRK4-mediated receptor phosphorylation. Furthermore, similar cAMP accumulation experiments performed with the D₁ DAR cluster mutants 1516 and 181920 revealed that the GRK4 effect was attenuated in the 181920 mutant and abolished with the 1516 mutant (Fig. 8). These data show that constitutive phosphorylation of the D₁ DAR by GRK4 results in a diminished ability of the receptor to stimulate cAMP production and that this effect is primarily mediated by phosphorylation of Thr428 and Ser431.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Effect of GRK4 on D1 receptor-stimulated cAMP accumulation. HEK293T cells were cotransfected with WT, T404 carboxyl truncation mutant, or mutT404 D1 receptor constructs (see Fig. 4 for nomenclature) with or without GRK4. Two days after transfection, the cells were challenged with the indicated doses of dopamine for 15 min and cAMP accumulation assays were performed as described under Materials and Methods. The data are expressed as a percentage of the maximum cAMP response produced by the indicated receptor construct coexpressed with empty vector. A, WT D1 receptor was coexpressed with empty vector or GRK4. A single experiment is shown, representative of four individual experiments. B, D1 receptor truncation T404 was coexpressed with empty vector or GRK4. A single experiment is shown that is representative of four individual experiments. C, D1 receptor mutant mutT404 was coexpressed with empty vector or GRK4. A single experiment is shown that is representative of four individual experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effect of cluster mutations on GRK4-mediated regulation of D1 receptor-stimulated cAMP accumulation. Cells were cotransfected with wild-type (WT) or cluster mutant (1516 or 181920) D1 receptor constructs (see Fig. 4 for nomenclature) with or without GRK4 as described in Fig. 7. The data are expressed as a percentage of the maximum cAMP response produced by the indicated receptor construct coexpressed with empty vector. A, wild-type (WT) D1 receptor was coexpressed with empty vector or GRK4. A single experiment is shown, representative of four individual experiments. B, D1 receptor cluster mutant 1516 was coexpressed with empty vector or GRK4. A single experiment is shown, representative of four individual experiments. C, D1 receptor cluster mutant 181920 was coexpressed with empty vector or GRK4. A single experiment is shown, representative of four individual experiments.

Because it seems that constitutive GRK4-mediated phosphorylation of the D₁ DAR produces desensitization of the receptor response, we wondered whether subsequent agonist treatment might produce even further desensitization. To test this, HEK293T cells were transiently transfected with the wild-type receptor, in the presence and absence of GRK4, and then pretreated with 10 µM dopamine for 1 h. Figure 9A shows that dopamine pretreatment of the cells transfected with the D₁ DAR and empty vector reduces the maximum cAMP response by 30 to 40%, in agreement with our previous findings (Kim et al., 2004). In cells cotransfected with GRK4, the maximum cAMP response is already diminished by approximately 50%, as shown previously in Figs. 7A and 8A; however, dopamine pretreatment produces a further loss of 30 to 40% compared with untreated cells (Fig. 9A). Thus, the degree of agonist-induced desensitization is similar in the absence or presence of GRK4 (Fig. 9B). It is noteworthy that GRK4 coexpression does not completely eliminate agonist-induced D₁ DAR phosphorylation (Fig. 1) suggesting that one or more of the functionally relevant sites phosphorylated by endogenous GRKs in these cells are not phosphorylated by GRK4. HEK293T cells have been reported to express GRKs 2, 3, 5, and 6 (Iwata et al., 2005; Kim et al., 2005), and we have shown that GRK2 and GRK3 can promote phosphorylation of the D₁ DAR (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 9. Agonist-induced desensitization of wild-type D1 receptor in the presence and absence of GRK4. HEK293T cells were transfected with wild-type (WT) D1 receptor in the absence (empty vector V) or presence of GRK4. Agonist-desensitization (Des) was assessed by incubating cells with 10 µM dopamine for 1 h, washing, and rechallenging the cells with the indicated doses of dopamine for 15 min. A, cAMP accumulation assay performed on cells transfected as indicated and performed as described under Materials and Methods. A representative experiment performed five times with similar results is shown. B, the percentage of desensitization for each transfection group was calculated by dividing the maximum cAMP produced by the desensitized cells (dashed lines in A) by the maximum cAMP produced by the corresponding control cells (solid lines in A), then subtracting this ratio from 1 and converting to a percentage. Percentages for each transfection group were calculated for five independent experiments and the average percentage desensitization ± S.E.M. are reported.

Effect of GRK4 on D₁ DAR Expression and Subcellular Location. We were next interested in investigating whether GRK4-mediated phosphorylation might affect the expression of the D₁ DAR. Our first approach was to measure the total complement of cellular receptors using the D₁-selective antagonist [³H]SCH-23390. Saturation binding assays were performed on membranes derived from HEK293T cells expressing the D₁ DAR in the absence or presence of GRK4 and maximum binding capacities (B_max) were determined. Figure 10 shows that cotransfection of the wild-type D₁ DAR with GRK4 results in a 25% reduction in total D₁ receptor expression (B_max). It is noteworthy that this effect by GRK4 was not observed with the 1516 mutant D₁ DAR; however, the 181920 mutant produced results similar to those observed with the wild-type receptor. These results agree well with those obtained with the cAMP accumulation assays (Fig. 8) and support the notion that Thr428 and Ser431 (mutated in the 1516 construct) are the functionally relevant GRK4 phosphorylation sites.

View larger version (18K): [in this window] [in a new window]   Fig. 10. Effect of GRK4 on the expression of the D1 receptor in HEK293T cells. The cells were transiently transfected with WT D1 receptor or the cluster mutants 1516 or 181920 (see Fig. 4 for nomenclature) in the absence (V) or presence of GRK4 (+4). After 2 days, the cells were harvested, membranes were prepared, [3H]SCH-23390 saturation binding assays were performed, and Bmax values were determined as described under Materials and Methods. Because transiently transfected cells were used to express each construct, the receptor binding data (Bmax values) are reported as the percentage of control as a result of the variability in exact receptor expression from experiment to experiment. Cells were transfected with each combination of expression constructs indicated for each individual experiment. Control receptor expression and affinities for each construct were not significantly different within an individual experiment. Ranges for all experiments are as follows: Bmax = 6 to 12 pmol receptor/mg of protein, and KD = 0.075 to 0.32 nM for [3H]SCH-23390. Maximum binding for WT and cluster mutant 181920 constructs coexpressed with GRK4 were found to be significantly reduced compared with corresponding control cells (*, p < 0.05, Student's t test, paired).

Because the reduction of total receptor expression in response to GRK4 was modest (25%) compared with the reduction in cAMP response (50%), we sought to determine whether phosphorylation of the D₁ DAR by GRK4 might additionally influence its location within the cell. Experiments were thus conducted using confocal fluorescence microscopy and a D₁-GFP fusion construct that enables the intracellular visualization of the D₁ DAR in real-time (Bermak et al., 2002). Figure 11, top, shows that under basal conditions, the D₁ DAR is primarily located in the plasma membrane, although some intracellular fluorescence is observed. Short-term treatment of the cells with dopamine promotes internalization of the D₁ DAR and an accumulation of intracellular fluorescence. Figure 11, bottom, shows that coexpression of GRK4 results in a substantial increase in the internalization of the D₁ DAR under basal conditions. In this case, the only observable effect of dopamine treatment is to promote a small further decrease in cell surface receptor expression. These results suggest that constitutive phosphorylation of the D₁ DAR by GRK4 promotes its sequestration from the cell surface under basal conditions.

View larger version (109K): [in this window] [in a new window]   Fig. 11. Confocal fluorescence microscopy of D1 receptor-GFP expression in the presence or absence of GRK4. HEK293T cells were transfected with wild-type D1 receptor-GFP construct and either empty vector or GRK4. Twenty-four hours after transfection, cells were plated on glass-bottomed 35-mm culture dishes, and confocal microscopy was performed using a Zeiss laser-scanning confocal microscope (LSM-510). Images were collected sequentially every 30 s after stimulation with 20 µM dopamine using single line excitation (488 nm). Shown are representative confocal microscopic images of D1 receptor-GFP fluorescence before dopamine stimulation (control) and after 720 s (12 min) of dopamine treatment. This experiment was performed three times with similar results.

View larger version (107K): [in this window] [in a new window]   Fig. 12. Effect of a dominant-negative mutant of -arrestin on dopamine-induced D1-GFP internalization in the absence or presence of GRK4. HEK293T cells were cotransfected with wild-type D1 receptor-GFP and a dominant-negative mutant of -arrestin1 (Krupnick et al., 1997) and either empty vector or GRK4. Twenty-four hours after transfection, cells were plated on 35 mm glass-bottom culture dishes, and confocal microscopy was performed on a Zeiss laser-scanning confocal microscope (LSM-510). Images were collected sequentially every 30 s after stimulation with 20 µM dopamine using single line excitation (488 nm). Shown are representative confocal microscopic images of D1 receptor-GFP fluorescence before dopamine stimulation (control) and after 720 s (12 min) of dopamine treatment. This experiment was performed three times with similar results.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have investigated the role and specificity of nonvisual GRK-mediated phosphorylation of the D₁ dopamine receptor. In agreement with previous findings (Tiberi et al., 1996), we found that GRK2 and GRK3 both enhanced agonist-induced phosphorylation of the D₁ receptor. In contrast, we did not observe consistent effects with either GRK5 or GRK6, suggesting that these GRKs may not play a major role in D₁ receptor phosphorylation. We were surprised to find that expression of GRK4 with the D₁ receptor resulted in enhanced receptor phosphorylation even in the absence of agonist activation. This constitutive phosphorylation of the receptor by GRK4 was found to be specific for the isoform of the kinase—the , , and isoforms were all inactive in this regard. As shown in Fig. 2, the GRK4 gene contains two cassette exons encoding 32 or 46 amino acids near the enzyme's amino and carboxyl termini, respectively, that can undergo alternative RNA splicing. Only the isoform of GRK4 contains both cassette exon sequences, suggesting that both regions are needed for constitutive phosphorylation of the D₁ receptor. The mechanism by which this occurs is not clear, although possibilities include a higher enzymatic activity of the isoform or specific interactions with a scaffold or adaptor protein that facilitates GRK4/D₁ receptor interactions. In this regard, similar results have been reported by Sallese et al. (1997), who found that only GRK4, but not GRK4, -, or -, was able to phosphorylate light-activated rhodopsin using a reconstitution system. This activity was found to correlate with the ability of GRK4 to interact with calmodulin, a property not exhibited by the other GRK4 isoforms (Sallese et al., 1997). It will therefore be of interest to determine whether calmodulin, or a related interacting protein, is involved in GRK4-mediated phosphorylation of the D₁ receptor.

Mutational analysis revealed that constitutive phosphorylation of the D₁ receptor by GRK4 was restricted to serine and threonine residues within the distal carboxyl terminus of the receptor. Two clusters of GRK4 phosphorylation sites were identified: the first consists of Thr428 and Ser431, whereas the second consists of Ser441, Ser445, and Thr446 (Fig. 4). Mutation of these residues— either singly, as clusters, all together, or via truncation of the distal end of the carboxyl terminus—reduced or eliminated constitutive phosphorylation by GRK4. This effect was most pronounced for the Thr428/Ser431 mutant, suggesting that these two residues constitute the majority of the GRK4 phosphorylation sites. It is noteworthy that close inspection of the receptor sequence immediately surrounding these residues reveals the existence of several acidic amino acids that are typically found near residues that are phosphorylated by GRKs (Palczewski et al., 1989; Onorato et al., 1991; Fredericks et al., 1996).

Constitutive phosphorylation of the D₁ receptor by GRK4 was found to be associated with desensitization of receptor-stimulated cAMP accumulation and internalization of the receptor in the absence of agonist stimulation. Simultaneous mutation of Thr428 and Ser431 abrogated the constitutive desensitization induced by GRK4 coexpression, supporting the notion that these residues comprise the functionally relevant GRK4 phosphorylation sites. These results suggest that phosphorylation by GRK4 may promote -arrestin/D₁ receptor association even in the absence of agonist activation. The fact that coexpression with a dominant-negative mutant of -arrestin blocked the effects of GRK4 on D₁ receptor internalization lends support to this hypothesis. It is conceivable that phosphorylation by GRK4 may also reduce D₁ receptor/G protein coupling through conformational alterations of the carboxyl terminus, although this remains to be tested. It is interesting that constitutive phosphorylation by GRK4 was found to reduce the total cellular expression levels of the D₁ receptor—an effect that was absent in the Thr428/Ser431 mutant. This observation is probably related to increased and prolonged internalization of the D₁ receptor in the presence of GRK4 leading to enhanced receptor down-regulation. Lamey et al. (2002) found that mutation of Ser431 in the D₁ receptor reduced agonist-induced down-regulation, supporting the notion that this residue is important for receptor trafficking.

Although constitutive phosphorylation of carboxyl terminal residues Thr428 and Ser431 by GRK4 promotes receptor desensitization and internalization, these are clearly not the sole GRK phosphorylation sites within the D₁ receptor. It is noteworthy that agonist pretreatment in GRK4 transfected cells results in further desensitization of the cAMP response, an effect that is associated with a marginal enhancement of receptor phosphorylation. Furthermore, D₁ receptor mutants that do not exhibit GRK4-mediated phosphorylation exhibit a robust phosphorylation response when treated with agonists. Finally, our previous data suggested the presence of functionally relevant GRK phosphorylation sites within the 3rd cytoplasmic loop of the receptor (Kim et al., 2004). Taken together, these observations suggest that the residues constitutively phosphorylated by GRK4 may constitute a subset of the total number of GRK phosphorylation sites within the D₁ receptor. Because we have shown that both GRK2 and GRK3 can phosphorylate the agonist-activated D₁ receptor, and because both of these GRKs are endogenously expressed in HEK293 cells, it would seem that GRK2 and/or GRK3 exhibit broader specificity for phosphorylating D₁ receptor residues than GRK4. Further mapping of the functionally relevant GRK2/3 phosphorylation sites within the D₁ receptor is currently in progress.

Our current results are highly relevant to recent research demonstrating that the GRK4 gene is linked to human essential hypertension (Felder et al., 2002; Zeng et al., 2004). Abnormalities in renal dopaminergic systems have long been suggested as precipitating mechanisms for hypertension (Zeng et al., 2004). For example, it has been shown that the D₁ receptor in the kidney, which normally acts to regulate Na⁺ transport, is functionally diminished. This loss of activity does not seem to be due to any alteration in receptor sequence and is specific for D₁ receptors that are expressed in renal tissue. Felder et al. (2002) have shown that GRK4 is expressed in the kidney and can promote D₁ receptor desensitization. Using isolated renal proximal tubule cells, these investigators showed that both GRK4 and D₁ receptors were coexpressed and that D₁ receptor stimulation of cAMP accumulation was diminished in renal proximal tubule cells from hypertensive patients compared with those from normotensive controls subjects. These results suggest that GRK4 may tonically dampen D₁ receptor signaling in renal tissue and that this effect may be increased in hypertensive subjects.

Indeed, the GRK4 gene locus, 4p16.3, is linked to hypertension, and it has been suggested that activating variants of GRK4 diminish D₁ receptor signaling in the kidney leading to increased blood pressure (Zeng et al., 2004). Three SNPs leading to altered GRK4 sequences (R65L, A142V, and A486V) have been investigated within the context of the isoform and have indeed been found to promote enhanced D₁ receptor desensitization (Felder et al., 2002). Although our current results strongly support a role for GRK4-mediated constitutive desensitization of the D₁ receptor in hypertension, we observed no effects on D₁ receptor phosphorylation by coexpressing the GRK4 SNPs R65L, A142V, and A486V (Fig. 3). One possible explanation for these results is the observed specificity of the GRK4 isoform for constitutively phosphorylating the D₁ receptor or possibly cell-type specificity for GRK4 isoform/D₁ receptor interactions. Therefore, it will be important to reinvestigate the identified SNPs within the context of the GRK4 isoform for their ability to enhance D₁ receptor phosphorylation.

Perhaps the most important observation of our present study is the demonstration of GRK-mediated phosphorylation of a GPCR in an agonist-independent fashion. Until now, GRK-mediated phosphorylation of GPCRs has been suggested to be a strictly agonist-dependent process that links the process of receptor activation to desensitization (Ferguson et al., 1996; Penela et al., 2003; Gainetdinov et al., 2004). The only notable exceptions to this rule have been GPCRs harboring mutations that render them constitutively active (Pei et al., 1994); however, the wild-type D₁ receptor does not exhibit constitutive activity (Charpentier et al., 1996). Our data thus expand the functionality of GRKs from kinases that phosphorylate only agonist-bound, activated receptors to kinases that are capable, under certain conditions, of phosphorylating additional receptor conformations (unoccupied, inactive receptors), resulting in profound modulation of the signaling potential for GPCRs. In the present example, tissue-specific (e.g., kidney) expression of GRK4 has the potential to dramatically alter the phosphorylation state, functionality and location of the unoccupied, inactive D₁ receptor. These observations may provide a rationale for the presence of multiple GRK isoforms whereby the signaling potential for a given GPCR can be modulated, both in the presence and absence of agonist, by the particular repertoire of kinases coexpressed within a specific cellular environment.

	Acknowledgements

 
We thank the National Institute of Neurological Disorders and Stroke DNA sequencing facility for generating all sequence data. Wewould also like to thank Dr. Vanitha Ramakrishnan for the HEK293tsa201 cells, Dr. Qun-Yong Zhou (University of California, Irvine, CA) for the rat D₁-GFP construct, Dr. Richard T. Premont (Duke University Medical Center, Durham, NC) for the GRK4 isotypes, and Dr. Jeffrey L. Benovic (Kimmel Cancer Center, Philadelphia, PA) for the GRK2, GRK3, and dominant-negative -arrestin minigene (319-418) constructs.

	Footnotes

 
This research was supported in part by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health. M.L.R. was supported by a NINDS Competitive Fellowship.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.019901.

ABBREVIATIONS: GPCR, G protein-coupled receptor; ARK, -adrenergic receptor kinase; GRK, G protein-coupled receptor kinase; DAR, dopamine receptor; HEK, human embryonic kidney; SNP, single-nucleotide polymorphism; GFP, green fluorescent protein; SCH-23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; Ro-20-1724, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one; PVDF, polyvinylidene difluoride; DMEM, Dulbecco's modified essential medium; EBSS, Earle's balanced salt solution; MOPS, 3-(N-morpholino)-propanesulfonic acid; TBST, Tris-buffered saline containing 0.05% Tween 20; WT, wild type; V, vector.

Address correspondence to: Dr. David R. Sibley, Molecular Neuropharmacology Section, NINDS/NIH, 5625 Fishers Lane, Room 4S-04, MSC 9405, Bethesda, MD 20892-9405. E-mail: sibley{at}helix.nih.gov

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