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G Protein-coupled Receptor Kinase Regulates Dopamine D3 Receptor Signaling by Modulating the Stability of a Receptor-Filamin-β-Arrestin Complex

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m408901200

ISSN

1083-351X

Autores

Kyeong-Man Kim, Raul R. Gainetdinov, Stéphane A. Laporte, Marc G. Caron, Larry S. Barak,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

In addition to its postsynaptic role, the dopamine D3 receptor (D3R) serves as a presynaptic autoreceptor, where it provides continuous feedback regulation of dopamine release at nerve terminals for processes as diverse as emotional tone and locomotion. D3R signaling ability is supported by an association with filamin (actin-binding protein 280), which localizes the receptor with G proteins in plasma membrane lipid rafts but is not appreciably antagonized in a classical sense by the ligand-mediated activation of G protein-coupled receptor kinases (GRKs) and β-arrestins. In this study, we investigate GRK-mediated regulation of D3R·filamin complex stability and its effect on D3 R protein ·G signaling potential. Studies in HEK-293 cells show that in the absence of agonist the D3R immunoprecipitates in a complex containing both filamin A and β-arrestin2. Moreover, the filamin directly interacts with β-arrestin2 as assessed by immunoprecipitation and yeast two-hybrid studies. With reductions in basal GRK2/3 activity, an increase in the basal association of filamin A and β-arrestin2 with D3R is observed. Conversely, increases in the basal GRK2/3 activity result in a reduction in the interaction between the D3R and filamin but a relative increase in the agonist-mediated interaction between β-arrestin2 and the D3R. Our data suggest that the D3R, filamin A, and β-arrestin form a signaling complex that is destabilized by agonist- or expression-mediated increases in GRK2/3 activity. These findings provide a novel GRK-based mechanism for regulating D3R signaling potential and provide insight for interpreting D3R autoreceptor behavior. In addition to its postsynaptic role, the dopamine D3 receptor (D3R) serves as a presynaptic autoreceptor, where it provides continuous feedback regulation of dopamine release at nerve terminals for processes as diverse as emotional tone and locomotion. D3R signaling ability is supported by an association with filamin (actin-binding protein 280), which localizes the receptor with G proteins in plasma membrane lipid rafts but is not appreciably antagonized in a classical sense by the ligand-mediated activation of G protein-coupled receptor kinases (GRKs) and β-arrestins. In this study, we investigate GRK-mediated regulation of D3R·filamin complex stability and its effect on D3 R protein ·G signaling potential. Studies in HEK-293 cells show that in the absence of agonist the D3R immunoprecipitates in a complex containing both filamin A and β-arrestin2. Moreover, the filamin directly interacts with β-arrestin2 as assessed by immunoprecipitation and yeast two-hybrid studies. With reductions in basal GRK2/3 activity, an increase in the basal association of filamin A and β-arrestin2 with D3R is observed. Conversely, increases in the basal GRK2/3 activity result in a reduction in the interaction between the D3R and filamin but a relative increase in the agonist-mediated interaction between β-arrestin2 and the D3R. Our data suggest that the D3R, filamin A, and β-arrestin form a signaling complex that is destabilized by agonist- or expression-mediated increases in GRK2/3 activity. These findings provide a novel GRK-based mechanism for regulating D3R signaling potential and provide insight for interpreting D3R autoreceptor behavior. The D3 dopamine receptor (D3R), 1The abbreviations used are: D3R, dopamine D3 receptor; CCP, clathrin-coated pit; D2R, dopamine D2 receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; DA, dopamine; GTPγS, guanosine 5′-3-O-(thio)triphosphate; β2AR, β2-adrenergic receptor; HA, hemagglutinin; GFP, green fluorescent protein. in addition to serving in a classical postsynaptic sensory role, is also a presynaptic autoreceptor that is predominantly expressed in parts of the brain controlling behavior, including that involved with emotion and movement (1.Joseph J.D. Wang Y.M. Miles P.R. Budygin E.A. Picetti R. Gainetdinov R.R. Caron M.G. Wightman R.M. Neuroscience. 2002; 112: 39-49Crossref PubMed Scopus (143) Google Scholar). The D3R is homologous to four other dopamine receptor subtypes (2.Missale C. Nash S.R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2773) Google Scholar) but is closest in expression and function to the D2R, (1.Joseph J.D. Wang Y.M. Miles P.R. Budygin E.A. Picetti R. Gainetdinov R.R. Caron M.G. Wightman R.M. Neuroscience. 2002; 112: 39-49Crossref PubMed Scopus (143) Google Scholar, 3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The D2 and D3 autoreceptors have overlapping distributions in the brain (2.Missale C. Nash S.R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2773) Google Scholar, 4.Diaz J. Pilon C. Le Foll B. Gros C. Triller A. Schwartz J.C. Sokoloff P. J. Neurosci. 2000; 20: 8677-8684Crossref PubMed Google Scholar, 5.Gurevich E.V. Joyce J.N. Neuropsychopharmacology. 1999; 20: 60-80Crossref PubMed Scopus (388) Google Scholar), where they regulate presynaptic release of dopamine (DA) by at least three different mechanisms: by affecting rates of intracellular DA synthesis, by mediating vesicular secretion of DA at the plasma membrane, and by modulating reuptake of dopamine at plasma membrane DA transporters (1.Joseph J.D. Wang Y.M. Miles P.R. Budygin E.A. Picetti R. Gainetdinov R.R. Caron M.G. Wightman R.M. Neuroscience. 2002; 112: 39-49Crossref PubMed Scopus (143) Google Scholar, 6.Jones S.R. Gainetdinov R.R. Hu X.T. Cooper D.C. Wightman R.M. White F.J. Caron M.G. Nat. Neurosci. 1999; 2: 649-655Crossref PubMed Scopus (206) Google Scholar). D3R has a higher affinity for DA than D2R, and experiments with D3R knock-out mice suggest that in the striatum/nucleus acumbens the D3R may regulate DA release at lower DA concentrations than D2R by modulating DA secretion rates rather than DA synthesis or DA transporter activity (1.Joseph J.D. Wang Y.M. Miles P.R. Budygin E.A. Picetti R. Gainetdinov R.R. Caron M.G. Wightman R.M. Neuroscience. 2002; 112: 39-49Crossref PubMed Scopus (143) Google Scholar). The G protein-mediated signaling of G protein-coupled receptors (GPCR) in general and D2 and D3Rs in particular should terminate from arrestin protein binding to receptor intracellular loop or C-tail residues that are GPCR kinase (GRK)-phosphorylated (7.Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar, 8.Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 9.Kim O.J. Gardner B.R. Williams D.B. Marinec P.S. Cabrera D.M. Peters J.D. Mak C.C. Kim K.M. Sibley D.R. J. Biol. Chem. 2004; 279: 7999-8010Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The subsequent trafficking of arrestin-receptor complexes to clathrin-rich regions of plasma membrane and clathrin-coated pits (CCPs) depends on the extent of GRK phosphorylation (10.Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 19452-19460Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) and results in a sustained blockade of G protein coupling, the establishment of arrestin-dependent non-G protein-mediated signaling, and the endocytosis of arrestin-receptor complexes (7.Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar, 8.Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 11.McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). CCP-initiated recycling of dephosphorylated receptors contributes to relief of the blockade in G protein signaling and a reinitiation of membrane signaling competence (12.Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 13.Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Autoreceptors that function as permanent sensors of extracellular neurotransmitter concentrations may require alternative mechanisms to regulate the transition between the classical G protein signaling and desensitized states observed for receptors with more conventional signaling responsibilities. In fact, although D3Rs can be induced to internalize to a small degree, the large majority of D3Rs remain at the plasma membrane and fail to internalize appreciably in CCPs after brief periods of DA stimulation (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The D3R and D2R undergo remarkably different degrees of arrestin-directed trafficking to CCPs (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The molecular determinants including GRK phosphorylation sites underlying CCP association are confined to their relatively large third intracellular loops as demonstrated by experiments in which the loops were interchanged between receptors (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In contrast to this post signaling behavior, the D3R and D2R share a requirement for binding lipid raft-associated filamin (actin-binding protein 280) in order to couple efficiently to G proteins (14.Lin R. Karpa K. Kabbani N. Goldman-Rakic P. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5258-5263Crossref PubMed Scopus (170) Google Scholar, 15.Li M. Li C. Weingarten P. Bunzow J.R. Grandy D.K. Zhou Q.Y. Biochem. Pharmacol. 2002; 63: 859-863Crossref PubMed Scopus (26) Google Scholar, 16.Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar). Interestingly, these associations are also mediated by third intracellular loop residues (14.Lin R. Karpa K. Kabbani N. Goldman-Rakic P. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5258-5263Crossref PubMed Scopus (170) Google Scholar, 15.Li M. Li C. Weingarten P. Bunzow J.R. Grandy D.K. Zhou Q.Y. Biochem. Pharmacol. 2002; 63: 859-863Crossref PubMed Scopus (26) Google Scholar, 16.Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar). Filamin A also binds other GPCRs, including metabotropic glutamate receptors type 7 (17.Enz R. FEBS Lett. 2002; 514: 184-188Crossref PubMed Scopus (60) Google Scholar), calcitonin receptors (18.Seck T. Baron R. Horne W.C. J. Biol. Chem. 2003; 278: 10408-10416Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), and μ-opioid receptors (19.Onoprishvili I. Andria M.L. Kramer H.K. Ancevska-Taneva N. Hiller J.M. Simon E.J. Mol. Pharmacol. 2003; 64: 1092-1100Crossref PubMed Scopus (89) Google Scholar), suggesting that filamin may be an integral component for regulating the stability of receptor-G protein signaling complexes. The small extent of DA-mediated D3R endocytosis (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) suggests that the repetitive cycles of ligand-mediated GRK phosphorylation followed by intracellular dephosphorylation common to many other GPCRs are not critical for regulating D3R signaling (13.Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). However, GRK knock-out mice have revealed that receptors underlying dopaminergic-mediated locomotion do become supersensitive to ligand in the absence of some specific GRKs (20.Gainetdinov R.R. Premont R.T. Bohn L.M. Lefkowitz R.J. Caron M.G. Annu. Rev. Neurosci. 2004; 27: 107-144Crossref PubMed Scopus (684) Google Scholar). Thus, significant GRK modulation of D3R behavior may be highly likely despite the arrestin-trafficking results indicating otherwise. Here we show that D3R, β-arrestin, and filamin form a basal signaling complex. The elevation of cellular GRK activity either by ligand addition or increases in the absolute amount of intracellular GRKs decreases the stability of this complex and results in a dramatic reduction in the G protein signaling potential of the D3R. These findings suggest how the D3R, as an autoreceptor, could rapidly desensitize and resensitize in order to provide fast feedback on short term fluctuations in DA concentration. Materials—Human embryonic kidney cells (HEK-293) and COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA). Cell culture reagents were from Cellgro (Herndon, VA) or Invitrogen. A human melanoma cell line (M2) that does not express filamin A endogenously and the A7 cell line, an M2 subclone that has been stably transfected with filamin A cDNA, are described in previous studies (21.Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar, 22.Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (376) Google Scholar). [32P]Orthophosphate, [3H]sulpiride, [3H]adenine, [35S]GTPγS, and [14C]cAMP were from PerkinElmer Life Sciences, and [3H]spiperone was from Amersham Biosciences. Anti-FLAG M2 antibody and horseradish peroxidase-labeled anti-mouse or anti-rabbit antibodies were from Sigma, anti-HA antibody was from Roche Applied Science, and anti-filamin A antibody was from Research Diagnostics Inc. (Flanders, NJ). Antibodies for β-arrestins and GRK2/GRK3 were provided by Drs. R. Lefkowitz and R. Premont (Duke University Medical Center), respectively. Cell Culture and Transfection—HEK-293 cells or COS-7 cells were cultured in minimal essential medium or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin in a humidified atmosphere containing 5% CO2. Transfections were carried out by the calcium phosphate precipitation method or using Lipofectamine (Invitrogen). Cells were used for the experiments 36 h following transfection to 60 h post-transfection for the cAMP assay. M2 and A7 cells were grown in minimal essential medium supplemented with 8% newborn calf serum and 2% fetal bovine serum. A7 cells were maintained in a medium containing G418 (500 μg/ml; Research Products International Corp., Mt. Prospect, IL). Transfections of M2 and A7 cells were conducted by electroporation at a setting of 1000 microfarads, 240–320 V using a Gene Pulser® (Bio-Rad) in a Cytomix buffer consisting of 25 mm HEPES, 120 mm KCl, 10 mm KH2PO4, 0.15 mm CaCl2, 5 mm MgCl2, 2 mm EGTA, pH 7.6. Generation of Plasmid Constructs—Wild type human D2R and D3R in mammalian expression vector pCMV5 were previously described (23.Robinson S.W. Caron M.G. J. Neurochem. 1996; 67: 212-219Crossref PubMed Scopus (38) Google Scholar). Both short and long isoforms of dopamine D2R (D2SR and D2LR) were tagged at the amino terminus with the M2-FLAG epitope, and D3R was tagged at the amino terminus with the HA epitope or with the M2-FLAG epitope (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Expression constructs for GRKs 2 and 3 in pRK5, β-arrestins 1 and 2 in pCMV5, β-arrestin2-GFP in pEGFP, GRK2-K220R in pCDNA 1.1, and dynamin K44A in pRK5 have been described previously (24.Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 25.Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (202) Google Scholar, 26.Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). D3R-S309A was constructed by site-directed mutagenesis, and filamin A in pCDNA 3.0 was provided by Dr. Y. Ohta (Harvard University). Caine AC-V (adenylyl cyclase V) cDNA was from Dr. Y. Ishikawa (Columbia University). Yeast Two-hybrid Assay—The yeast two-hybrid vectors and reagents were purchased from Clontech (Palo Alto, CA), and library screening was conducted as previously described (27.Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar). The amino terminus of β-arrestin2 (amino acids 1–80) subcloned into the yeast vector, pAS2-1, that contains the DNA-binding domain was used as bait. The human kidney cDNA library (100 μg) constructed in pACT2 and the bait plasmids were transformed into Saccharomyces cerevisiae strain Y187 and transformants were selected on artificial medium lacking tryptophan, leucine, and histidine. After a 4-day incubation at 30 °C, a β-galactosidase assay was conducted for His+ clones to eliminate false positives, and resulting His+/β-galactosidase+ clones were sequenced with an automated ABI DNA sequencer. Whole Cell Phosphorylation—HEK-293 cells were transfected with human β2-adrenergic receptor tagged at the amino terminus with the HA epitope (HA-β2AR), FLAG-D2R, or HA-D3R with or without GRKs. Cells were stimulated with 10 μm isoproterenol or DA for 5 min. Detailed procedures are described in our previous studies (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 25.Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (202) Google Scholar). The amount of receptor in each sample was determined by saturation binding with 3 nm [3H]spiperone. Protein concentration was assessed using the Bio-Rad DC protein assay. Phosphorylated receptors were quantitated using a PhosphorImager or were visualized by exposure to autoradiography film. Photoaffinity Labeling—Photoaffinity labeling of the D2R and D3R was carried out as previously described (28.Amlaiky N. Kilpatrick B.F. Caron M.G. FEBS Lett. 1984; 176: 436-440Crossref PubMed Scopus (18) Google Scholar). Confocal Microscopy—For the β-arrestin translocation assays, HEK-293 cells were transfected with β-arrestin2-GFP (24.Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar) and D3R with or without GRK3. One day after transfection, cells were seeded onto 35-mm dishes containing a centered, 1-cm well formed from a glass coverslip sealed hole in the plastic (confocal dishes) and allowed to recover 1 day. Cells were incubated with 2 ml of minimal essential medium containing 20 mm HEPES, pH 7.4, and viewed on a Zeiss laser-scanning confocal microscope. Immunoprecipitation—COS-7 or HEK-293 cells were transfected using Lipofectamine or the calcium phosphate precipitation method, respectively. After 48 h, cells were lysed in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) on the rotation wheel for 1 h at 4 °C. Supernatants were mixed with 35 μl of 50% slurry of FLAG-agarose beads (Sigma) between 2 and 3 h on the rotation wheel. Beads were washed with washing buffer (50 mm Tris, pH 7.4, 137 mm NaCl, 10% glycerol, 1% Nonidet P-40) three times for 10 min each. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted. GTPγS Binding—Membrane proteins were prepared in 20 mm HEPES, pH 7.4, 0.1 mm EDTA. For GTPγS binding, membrane proteins were incubated in 125 mm HEPES buffer containing 1 mm GDP and 100 nm [35S]GTPγS, incubated for 2 h at 30 °C, filtered on a GF/B filter, and counted with a liquid scintillation counter. The coupling efficiency between receptor and G protein was determined as fmol of Gα accumulated for 2 h at 30 °C/fmol of receptor added. Receptor expression levels were closely matched between experimental groups. Measurement of Cyclic AMP—Whole cell cyclic AMP accumulation was measured in HEK-293 cells by column chromatography as described in Ref. 23.Robinson S.W. Caron M.G. J. Neurochem. 1996; 67: 212-219Crossref PubMed Scopus (38) Google Scholar. Dopamine D3 Receptor Shows a Relatively Heavy Constitutive Phosphorylation—As shown in Fig. 1A (right), after radio-labeling of HEK-293 cells using [125I]NAPS (N-(p-aminophenethy)spiperone), D3Rs resolve into three different bands upon analysis by SDS-PAGE: a major band between 60 and 80 kDa, a minor band between 40 and 50 kDa, and a third band at 25 kDa, which may represent incompletely processed receptor protein. It is apparent from Fig. 1B that the total phosphorylation of the D3R in HEK-293 cells does not appreciably increase after 5 min of agonist exposure (pre- and postexposure results varied by less than 5%). We have previously demonstrated a similar basal relative lack of change in phosphorylation of the D3R to endogenous and overexpressed GRK2 in HEK cells at 10 min (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In contrast, the basal phosphorylation of the D3R was 5 times higher than the basal level of phosphorylation of a comparable amount of β2AR protein, whose phosphorylation also increased by more than 3-fold in the presence of isoproterenol (Fig. 1B, leftmost lanes). Moreover, the relatively high basal phosphorylation of the D3R and its lack of agonist-induced phosphorylation can be reversed by exchanging its third intracellular loop for the third intracellular loop of the D2R (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). These results indicate that D3R undergoes a marginal change in total phosphorylation in response to agonist stimulation of GRK activity as opposed to the 50% agonist-mediated increases in phosphorylation apparent for the D2LR (Fig. 1B, right panel), which was used as a positive control. Dopamine D3 Receptor Is Associated Basally with Arrestin— GRK-mediated receptor phosphorylation changes GPCR affinity for arrestins. Since the phosphorylation status of the D3Rin the absence of agonist was unexpectedly high compared with that of β2AR or D2R, the interaction between the D3R and β-arrestin was assessed for the basal state. A Western blot for β-arrestins isolated from D3R immunoprecipitates of COS-7 cells shows that the D3R associates with β-arrestin2 under basal conditions (Fig. 1C, lane 2). Agonist exposure does not significantly change the degree of this association (Fig. 1C, lanes 2 and 3). A negligible agonist-induced D3R phosphorylation is consistent with both the observed lack of change in the co-immunoprecipitation experiments (Fig. 1C, lane 3), and the absence of translocation of β-arrestins to D3Rs at 5 min after agonist treatment (Fig. 1D, upper panel). Exogenous GRK Expression Reduces the Basal Localization of the D3R with β-Arrestin while Facilitating Agonist-induced β-Arrestin Recruitment—We have observed both in COS-7 and HEK-293 cells that DA treatment does not augment the basal association of D3R with β-arrestin. However, an enhancement of GRK activity in these cells resulting from transfection of a plasmid encoding GRK noticeably increased the relative degree of agonist-dependent association of β-arrestins with D3R in clathrin-coated pits as observed by microscopy (Fig. 1D, lower panel). A corresponding immunoprecipitation experiment to determine the association of β-arrestin with the D3R in the presence of transfected GRK3 and DA treatment is shown in Fig. 2A (lanes 2 and 3). The results are consistent with the microscopic observations showing that arrestin translocation is observable in coated pits under these conditions (Fig. 1D, lower panels). The co-expression of GRKs appears to decrease the basal association of β-arrestins with D3Rs. This is evident from the Western blots showing the amounts of β-arrestin1 and -2 that were immunoprecipitated with the FLAG-tagged D3R in the absence of agonist (Fig. 2B, lane 2 versus lane 3 and lane 4 versus lane 5). We observed above that agonist can drive D3Rs to CCPs in the presence of elevated GRKs. To show that a similar behavior occurs under basal conditions, we trapped D3Rs in CCPs using the endocytosis inhibitor dynamin1-K44A. As shown in Fig. 2C, dynamin1-K44A alone did not have any effect on the subcellular localization of β-arrestin2-GFP. However, co-expression of GRK2 or GRK3 caused the accumulation of β-arrestin2-GFP at the plasma membrane. This accumulation of β-arrestin2-GFP is qualitatively similar to what we have previously reported for 10 μm dopamine-stimulated D3R in the presence of dynamin1-K44A (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). These results suggest that GRKs change the relative compartmentalization of the D3R with β-arrestins when agonist is absent either by reducing the complement of β-arrestins prebound to the D3R or shifting the equilibrium distribution of β-arrestin/D3R toward CCPs. Consequently, other proteins that are also basally associated with D3R may be affected by changes in GRK activity. β-Arrestin2 Interacts with the D3R-associated Filamin—The agonist-independent localization of β-arrestins with D3Rs suggests that proteins basally binding D3Rs are probable β-arrestin binding partners. This was tested in COS-7 cells for filamin A, which is localized in plasma membrane lipid rafts and is known to be important for the regulation of D3R signaling in cells (15.Li M. Li C. Weingarten P. Bunzow J.R. Grandy D.K. Zhou Q.Y. Biochem. Pharmacol. 2002; 63: 859-863Crossref PubMed Scopus (26) Google Scholar). COS-7 cells in comparison with other cell lines, such as HEK-293 cells, express relatively low levels of endogenous β-arrestins and thus provide a better background in which to assess transfected arrestin interactions by immunological methods (29.Menard L. Ferguson S.S. Zhang J. Lin F.T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (214) Google Scholar). An association under basal conditions between β-arrestin2 and filamin A was confirmed by Western blot of COS-7 cell immunoprecipitates obtained using FLAG-tagged β-arrestin2 and anti-FLAG antibodies (Fig. 3A). There are three human filamin isotypes (A, B, and C). They are ∼70% homologous and structurally similar, consisting of multiple (twenty-four) Ig-like repeats that extend through their C termini (30.van der Flier A. Sonnenberg A. Biochim. Biophys. Acta. 2001; 1538: 99-117Crossref PubMed Scopus (335) Google Scholar). Their degree of homology and functional redundancy (30.van der Flier A. Sonnenberg A. Biochim. Biophys. Acta. 2001; 1538: 99-117Crossref PubMed Scopus (335) Google Scholar) suggests they all are potential β-arrestin binding partners. Indeed, a yeast two-hybrid library screen in an attempt to identify β-arrestin2-interacting proteins using the first 80 amino acids of the β-arrestin2 N terminus as bait yielded two colonies containing the distal third of human filamin C. The ability of full-length β-arrestin2 to also interact with this filamin segment is shown in Fig. 3B. As observed above, the basal association of β-arrestin with filamin suggests that filamin may also be subject to GRK regulation. If so, D3R signaling could be dramatically affected by changes in GRK activity directed toward destabilizing filamin/receptor association. To investigate this question, we began by studying the general relationship between filamin and D3R signaling in cells that do not normally express filamin A. Filamin Expression Regulates the Coupling of the D3R to G Protein—The importance of filamin to D3R second messenger signaling can be established using M2 cells, which normally do not express filamin A (15.Li M. Li C. Weingarten P. Bunzow J.R. Grandy D.K. Zhou Q.Y. Biochem. Pharmacol. 2002; 63: 859-863Crossref PubMed Scopus (26) Google Scholar, 16.Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar). First, we are able to transfect M2 cells with quantitatively similar amounts of filamin A as contained in A7 cells (Fig. 4A). Although the total amounts of expressed immunoreactive filamin in the cell populations are similar, immunostaining and GFP co-transfection demonstrated that only one-third to one-half of the M2 cells are transfected and the efficiency of co-transfection, although appreciable, is less than 100% (data not shown). We investigated the relationship between filamin A and G protein signaling of the D3R in M2 cells by measuring GTPγS binding in the presence of increasing concentrations of DA (Fig. 4B). In order to improve the sensitivity of the assay, GTPγS binding was measured in M2 and control A7 cells transfected with the α subunit of Go protein (Gαo) in addition to D3R. With coupling efficiency defined as the number of GTPγS molecules bound to the α subunit of G protein per receptor molecule, the basal G protein coupling of the D3R was 9.4 ± 0.4% in M2 cells and 19.2 ± 1.7% in the control A7 cells, which express filamin A (Fig. 4B) (21.Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar). Co-expression of filamin A in M2 cells (M2-Filamin A) increased G protein coupling of D3R to levels more nearly equivalent to those observed in A7 cells (Fig. 4B, middle curve). D3R-S309A, a D3R mutant that has a point mutation in an amino acid important for filamin A binding (15.Li M. Li C. Weingarten P. Bunzow J.R. Grandy D.K. Zhou Q.Y. Biochem. Pharmacol. 2002; 63: 859-863Crossref PubMed Scopus (26) Google Scholar), exhibited reduced coupling efficiency (Fig. 4C). These results indicate a role for filamin A in D3R-G protein signaling and suggest that alteration of filamin A/D3R association would be one means to rapidly change D3R signaling potential. Agonist Treatment Reduces the Association between the D3R and Filamin A—Filamin A interacts with a motif in the D3R third cytoplasmic loop (14.Lin R. Karpa K. Kabbani N. Goldman-Rakic P. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5258-5263Crossref PubMed Scopus (170) Google Scholar, 16.Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar), and a decrease in the cellular content of filamin A reduces, but does not totally prevent, D3R signaling (Fig. 4, B and C). As such, receptors may retain the capacity to bind arrestins in the absence of filamin, and this is demonstrated by the immunoblot in Fig. 4D, where β-arrestin2 co-immunoprecipitated with FLAG-D3R in M2 cells. However, a dynamic reduction in the interaction between D3R and filamin A could provide a means to desensitize the receptor. We therefore tested whether agonist treatment disrupts the interaction between the D3R and filamin A. As shown in Fig. 4E by immunoprecipitation of FLAG-tagged D3R and immunoblotting for filamin A, the association of D3R with filamin A is rapidly reduced at 5 min to 64.0 ± 15.7% of control by DA treatment (30 nm). Exogenous GRK Expression Reduces the Basal Association between D3R and Filamin A—GRK activity increases secondarily to agonist activation of GPCRs. Therefore, we tested whether the ability of agonists to dissociate filamin A-receptor complexes could be reproduced by enhancing GRK activity without exposing receptors to agonist. In COS-7 cells, the co-expression of GRK2 or GRK3 reduces the basal interaction between D3R and filamin A (Fig. 5A, upper panel, compare lane 3 versus lane 4 and both lanes 3 and 4 versus lane 2) as demonstrated by immunoprecipitation of the FLAG-tagged receptor and immunoblotting for filamin A. Additionally, as discussed in Fig. 2B, GRK co-expression reduces the basal association of D3R with β-arrestins (Fig. 5A, lower panel, compare lane 3 and lane 4 and lane 3 versus lane 2). In contrast, a kinase-dead, dominant negative GRK2 (GRK2-K220R) enhances the basal association of the D3R with filamin A and that of the D3R with β-arrestin (Fig. 5B, compare lane 2 versus lane 3). These effects of GRK activity on the interactions between the D3R, filamin A, and β-arrestin were not direct results of GRK2 phosphorylation of filamin A. In other words, purified filamin A was not phosphorylated by purified GRK2 (data not shown). GRK Expression Reduces D3R Signaling—Since an association between the D3R and filamin A is needed for efficient G protein coupling, effects of GRK expression on the G protein coupling and the inhibition of cAMP production were tested. As expected from the results in Fig. 5, A and B, GRK overexpression significantly reduced both the basal and agonist-induced G protein coupling of the D3R as assessed by GTPγS binding (Fig. 5C). The GTPγS results indicate that second messenger production should be inhibited less well with excess GRK expression (i.e. the D3R-mediated decrease in cellular cAMP content should be smaller). To test the effects of GRKs on the ability of the D3R to inhibit cAMP production, the cellular cAMP was first raised by forskolin stimulation, and the D3R-mediated decrease in cellular cAMP was determined with increasing concentrations of quinpirole in cells transiently expressing GRK2 or GRK3. The dose-response curves in the presence of added kinase exhibit a decrease in Vmax and an increase in IC50 (i.e. a shift of the curve to the right) (Fig. 5D). The IC50 (nm) and the maximal inhibition (%) for the control (0.31 ± 0.09 nm and 70 ± 7% (p < 0.05)) is shifted to 0.58 ± 0.15 nm and 55 ± 3% for GRK2 and to 0.53 ± 0.01 nm and 49 ± 3% (p < 0.05) for GRK3. Recent studies on the distribution of D3R protein in the brain show that it is expressed on virtually all dopaminergic neurons (4.Diaz J. Pilon C. Le Foll B. Gros C. Triller A. Schwartz J.C. Sokoloff P. J. Neurosci. 2000; 20: 8677-8684Crossref PubMed Google Scholar, 5.Gurevich E.V. Joyce J.N. Neuropsychopharmacology. 1999; 20: 60-80Crossref PubMed Scopus (388) Google Scholar), strongly supporting the idea of the D3R as an autoreceptor. In agreement with this autoreceptor hypothesis, only a negligible fraction of plasma membrane D3R internalizes as a result of persistent DA stimulation, even with increased GRK activity, so that the plasma membrane population of D3Rs remains relatively constant (3.Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Therefore, GRK/β-arrestin-directed receptor internalization in CCPs plays a relatively minor role in the short term, agonist-mediated D3R desensitization process, whereas GRK regulation of D3R plasma membrane compartmentalization may play a more significant role. Our studies indicate that GRKs serve as rheostats for adjusting the agonist-mediated G protein signaling potential of D3Rs that are in basal conformations. The D3R possesses an extensive third intracellular loop that contains multiple, putative GRK phosphorylation sites surrounding and dispersed throughout the filamin binding domain (14.Lin R. Karpa K. Kabbani N. Goldman-Rakic P. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5258-5263Crossref PubMed Scopus (170) Google Scholar, 16.Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar). Our findings indicate that basal GRK phosphorylation of these sites potentially reduces D3R binding to filamin and greatly reduces G protein-mediated signaling. Thus, basal GRK activity through phosphorylation of the D3R third loop may simultaneously determine both the number of D3R signaling complexes in filamin-rich lipid rafts and the extent of their agonist-mediated signaling, since agonist binding appears to shift receptors away from an association with filamin to one with β-arrestin. In this novel GPCR desensitization model, basal GRK activity controls formation of D3R·β-arrestin·filamin complexes, raising the question as to why arrestins need to co-localize with receptors and filamin at this stage. A general and perhaps the most basic explanation may be that arrestins are co-localized because this region is where signaling occurs and where homologous desensitization should occur most expeditiously. An explanation for the D3R in particular may lie in the role D3Rs presumably fill as autoreceptors. In order to provide continuous monitoring of ligand concentrations, D3Rs should be more resistant to removal from the cell surface than receptors without this functional requirement. As such, the D3Rs exhibit relatively limited ability to remain associated with arrestins in comparison with other GPCRs, which is indicative of an intrinsically lower affinity. Therefore, from mass action considerations, the local concentration of arrestins plays a much greater role in sustaining D3R desensitization than it would for a receptor with intrinsically greater arrestin affinity. Moreover, induced changes in the local concentration of β-arrestins with D3Rs that are reciprocal to changes in GRK activity may provide a negative feedback for maintaining receptor signaling potential relatively constant. It would be interesting to test whether this novel model of GPCR desensitization will be applicable to other autoreceptor GPCRs that are involved in neurotransmitter concentration-dependent regulation of secretion. Overall, D3R signaling may be desensitized by at least two GRK-mediated mechanisms. Desensitization occurs with a reduction in G protein coupling due to an inability of filamin to localize receptors near G proteins. Alternatively, desensitization may occur from a potentially reversible, direct blockade of G protein coupling by arrestin/receptor binding. Only profound enhancements in GRK activity can marginally sustain a more classical β-arrestin/receptor desensitization picture involving clathrin-mediated receptor endocytosis. An observation indicating that a modulation of basal GRK activity could be therapeutically useful for DA-related illness stems from studies of GRK6 knock-out mice. These animals become hyperresponsive to DA stimulation (31.Gainetdinov R.R. Bohn L.M. Sotnikova T.D. Cyr M. Laakso A. Macrae A.D. Torres G.E. Kim K.M. Lefkowitz R.J. Caron M.G. Premont R.T. Neuron. 2003; 38: 291-303Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), indicating a role of GRK6 in regulation of postsynaptic D2-like receptors. Additionally, the GRK6 knock-out mouse striatal membranes display similarly increased GTPγS binding relative to wild type mouse controls, as do our control cells compared with cells expressing additional GRK2 or -3. Interestingly, we observed that GRK3-KO mice are hyposensitive to DA stimulations in locomotor assays (20.Gainetdinov R.R. Premont R.T. Bohn L.M. Lefkowitz R.J. Caron M.G. Annu. Rev. Neurosci. 2004; 27: 107-144Crossref PubMed Scopus (684) Google Scholar), a finding that would be consistent with a specific role of this GRK in the regulation of DA autoreceptor function. Thus, if a genetic reduction of GRK activity can enhance dopamine receptor signaling in vivo, then a corresponding type-specific GRK inhibitor/activator might facilitate responsiveness to suboptimal levels of endogenous dopamine in the treatment of movement disorders like Parkinson's disease or to other monoaminergic neurotransmitters such as serotonin in the treatment of mood disorders like depression. We thank Dr. Yasutaka Ohta for filamin reagents, plasmids, and cells and Dr. Allen Eckhardt for help in evaluating filamin A as a GRK2 substrate.

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