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Regulation of the Phosphorylation State of Rhodopsin by Dopamine

1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês

10.1074/jbc.273.13.7181

1083-351X

Igor P. Udovichenko, Alexandra C. Newton, David S. Williams,

Neuroscience and Neuropharmacology Research

G protein-coupled receptors (GPCRs) are regulated by kinases and phosphatases that control their phosphorylation state. Here, the possibility that the state of GPCR phosphorylation could be affected by paracrine input was explored. We show that dopamine increased the rate of dephosphorylation of rhodopsin, the light receptor, in intact frog retinas. Further, we found that rod outer segments from dopamine-treated retinas contained increased rhodopsin phosphatase activity, indicating that this effect of dopamine on rhodopsin was mediated by stimulation of rhodopsin phosphatase. Dopamine is a ubiquitous neuromodulator and, in the retina, is released from the inner cell layers. Thus, our results identify a pathway for feedback regulation of rhodopsin from the inner retina and illustrate the involvement of dopamine in paracrine regulation of the sensitivity of a GPCR. G protein-coupled receptors (GPCRs) are regulated by kinases and phosphatases that control their phosphorylation state. Here, the possibility that the state of GPCR phosphorylation could be affected by paracrine input was explored. We show that dopamine increased the rate of dephosphorylation of rhodopsin, the light receptor, in intact frog retinas. Further, we found that rod outer segments from dopamine-treated retinas contained increased rhodopsin phosphatase activity, indicating that this effect of dopamine on rhodopsin was mediated by stimulation of rhodopsin phosphatase. Dopamine is a ubiquitous neuromodulator and, in the retina, is released from the inner cell layers. Thus, our results identify a pathway for feedback regulation of rhodopsin from the inner retina and illustrate the involvement of dopamine in paracrine regulation of the sensitivity of a GPCR. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; ROS, rod outer segment; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; ROS, rod outer segment; PAGE, polyacrylamide gel electrophoresis. represent a widespread family of proteins that transduce a large variety of signals, such as light, odorants, hormones, and neurotransmitters. They have common structural elements, including seven transmembrane domains, and are regulated by many homologous mechanisms. Understanding these regulatory mechanisms is therefore a central question in signal transduction. Deactivation of a GPCR involves phosphorylation of the receptor, and its subsequent resensitization requires dephosphorylation. Accordingly, the light receptor, rhodopsin, undergoes light-dependent phosphorylation and must be subsequently dephosphorylated (1Kühn H. Nature. 1974; 250: 588-590Crossref PubMed Scopus (94) Google Scholar). The phosphorylation state of GPCRs is regulated typically by GPCR kinases (GRKs) and second messenger-regulated kinases, and, on the other hand, by phosphatases. GRKs preferentially phosphorylate agonist-occupied or activated GPCRs, whereas the second messenger-dependent kinases (cAMP-dependent protein kinase and protein kinase C) may phosphorylate nonactivated receptors (2Lefkowitz R.J. Cell. 1993; 74: 409-412Abstract Full Text PDF PubMed Scopus (399) Google Scholar, 3Hosey M.M. DebBurman S.K. Pals-Rylaarsdam R. Richardson R.M. Benovic J.L. Prog. Brain Res. 1996; 109: 169-179Crossref PubMed Google Scholar). Phosphatases that regulate GPCRs belong to the phosphatase 2A family or are dependent on Ca2+ (4Pitcher J.A. Payne E.S. Csortos C. DePaoli-Roach A.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8343-8347Crossref PubMed Scopus (159) Google Scholar, 5Palczewski K. Hargrave P.A. McDowell J.H. Ingebritsen T.S. Biochemistry. 1989; 28: 415-419Crossref PubMed Scopus (68) Google Scholar, 6Fowles C. Akhtar M. Cohen P. Biochemistry. 1989; 28: 9385-9391Crossref PubMed Scopus (54) Google Scholar, 7Kutuzov M.A. Bennett N. Eur. J. Biochem. 1996; 238: 613-622Crossref PubMed Scopus (38) Google Scholar, 8Vinos J. Jalink K. Hardy R.W. Britt S.G. Zuker C.S. Science. 1997; 277: 687-690Crossref PubMed Scopus (103) Google Scholar). The kinases and phosphatases that affect the phosphorylation of GPCRs may in turn be regulated. Most obviously, second messenger-regulated kinases may mediate input from different signal transduction pathways (9Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (956) Google Scholar, 10Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (849) Google Scholar, 11Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (190) Google Scholar). GRKs can also be regulated by other pathways (12Stoffel III, R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (44) Google Scholar). Rhodopsin kinase (GRK1) activity, for example, is inhibited by the Ca2+-binding protein, recoverin, when Ca2+levels are high (13Kawamura S. Nature. 1993; 362: 855-857Crossref PubMed Scopus (323) Google Scholar). β-Adrenergic receptor kinase (GRK2) and GRK5 are both phosphorylated by the second messenger kinase, protein kinase C, resulting in their activation (14Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (144) Google Scholar) and inactivation (16Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), respectively. Less is known about the dephosphorylation of GPCRs and the regulation of their phosphatases, although rhodopsin dephosphorylation appears to be affected by Ca2+ levels. Bovine rhodopsin can be dephosphorylated by a phosphatase 2A (5Palczewski K. Hargrave P.A. McDowell J.H. Ingebritsen T.S. Biochemistry. 1989; 28: 415-419Crossref PubMed Scopus (68) Google Scholar, 6Fowles C. Akhtar M. Cohen P. Biochemistry. 1989; 28: 9385-9391Crossref PubMed Scopus (54) Google Scholar) and by a Ca2+-sensitive phosphatase (7Kutuzov M.A. Bennett N. Eur. J. Biochem. 1996; 238: 613-622Crossref PubMed Scopus (38) Google Scholar), both of which are present in photoreceptor outer segments. In Drosophila, rhodopsin is dephosphorylated by the rdgC protein, which possesses a putative Ca2+-binding domain in addition to a phosphatase catalytic domain (8Vinos J. Jalink K. Hardy R.W. Britt S.G. Zuker C.S. Science. 1997; 277: 687-690Crossref PubMed Scopus (103) Google Scholar, 17Steele F.R. Washburn T. Rieger R. O'Tousa J.E. Cell. 1992; 69: 669-676Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Regulation of kinases and phosphatases thus provides upstream mechanisms for modulating GPCRs. The focus of the present study was on whether input from the inner retina could affect the phosphorylation state of rhodopsin. Such input could potentially originate from general light- or dark-adaptive signals or from a circadian oscillator. The most likely candidate for an intercellular messenger is the major catecholamine in retina, dopamine. Amacrine and interplexiform cells in the inner retina release dopamine in response to light and under the control of a circadian clock (18Iuvone P.M. Galli C.L. Garrison-Gund C.K. Neff N.H. Science. 1978; 202: 901-902Crossref PubMed Scopus (339) Google Scholar, 19Wirz-Justice A. Da Prada M. Reme C. Neurosci. Lett. 1984; 45: 21-25Crossref PubMed Scopus (118) Google Scholar). Photoreceptor cells possess dopamine receptors (20Brann M.R. Young III., W.S. Neurosci. Lett. 1986; 69: 221-226Crossref PubMed Scopus (41) Google Scholar, 21Muresan Z. Besharse J.C. J. Comp. Neurol. 1993; 331: 149-160Crossref PubMed Scopus (63) Google Scholar, 22Hillman D.W. Lin D. Burnside B. J. Neurochem. 1995; 64: 1326-1335Crossref PubMed Scopus (42) Google Scholar), and dopamine has been shown to influence retinomotor movements and phototransductive membrane shedding (23Djamgoz M.B. Wagner H.J. Neurochem. Int. 1992; 20: 139-191Crossref PubMed Scopus (229) Google Scholar, 24Burnside B. Nagle B. Prog. Retinal Res. 1988; 2: 67-109Crossref Scopus (122) Google Scholar, 25Besharse J.C. Iuvone P.M. Pierce M.E. Prog. Retinal Res. 1988; 7: 21-61Crossref Scopus (215) Google Scholar, 26Reme C. Wirz-Justice A. Aeberhard B. Rhyner A. Brain Res. 1984; 298: 99-106Crossref PubMed Scopus (17) Google Scholar). We demonstrate here that dopamine feedback to the photoreceptor cells affects the kinetics of rhodopsin dephosphorylation in intact frog retinas, indicating that the light receptor can be regulated by paracrine input. Dopamine hydrochloride, R(+)-SCH-23390 hydrochloride, and spiperone hydrochloride were purchased from Research Biochemicals International, Inc. [32P]Orthophosphoric acid (∼9,000 Ci/mmol) was from NEN Life Science Products. All other chemicals were reagent grade. Northern grass frogs (Rana pipiens) weighing 20–30 g were purchased from Carolina Biological Supply Co. and treated according to NIH and University of California at San Diego animal care guidelines. The procedure for incubation of retinas and analysis of rhodopsin phosphorylation followed that described previously (27Udovichenko I.P. Newton A.C. Williams D.S. J. Biol. Chem. 1997; 272: 7952-7959Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Retinas were removed from dark-adapted animals. Each intact retina was incubated under dim red light in 1 ml of amphibian culture medium (35 mm NaHCO3, 75 mmNaCl, 3 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 10 mm Na-HEPES, pH 7.3, 10 μg/ml phenol red, 1 mg/ml casamino acids, 10 mmβ-d(+)-glucose, 0.1 mg/ml Na-l-ascorbate, and 20 μCi/ml [32P]orthophosphate) for 2 h and then for 10 min with or without receptor ligands. One retina from each frog served as the experimental, and the other served as the control. Retinas were illuminated by a calibrated flash of light that photoexcited 6 ± 3 or 80 ± 6% of the rhodopsin (27Udovichenko I.P. Newton A.C. Williams D.S. J. Biol. Chem. 1997; 272: 7952-7959Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and then incubated up to 1 h under dim red light. All incubations were carried out at 22–23 °C. The specific32P incorporation into rhodopsin was determined after SDS-PAGE (12% acrylamide) by densitometry of Coomassie Blue-stained and radioactive (PhosphorImager) bands and expressed in relative units per constant amount of rhodopsin. Three methods were used for preparation of rhodopsin samples: Method 1, at the end of the incubation period, 250 μl of buffer A (20 mm sodium phosphate, 50 mm Tris-Cl, 10 mm EDTA, 10 mm EGTA, pH 7.3) was added to each retina. Rod outer segments (ROSs) were detached from the rest of the retina by vortexing for 10 s. After allowing the retina remnants to settle for 2 min on ice, the ROS-enriched fraction was removed and centrifuged for 30 s at 16,000 × g. The pellet (crude ROSs) was suspended in 100 μl of 1% SDS at room temperature and centrifuged (20,000 × g, 20 min, room temperature) to remove debris, before adding SDS-PAGE sample buffer; Method 2, the crude ROSs from Method 1 were purified further by sucrose gradient centrifugation (28Papermaster D.S. Dreyer W.J. Biochemistry. 1974; 13: 2438-2444Crossref PubMed Scopus (579) Google Scholar); and Method 3, reactions of rhodopsin phosphorylation in intact retinas were stopped by adding 250 μl of 50% trichloroacetic acid, and the total retinal proteins were subjected to SDS-PAGE. In all three methods, each rhodopsin fraction from an individual retina in SDS-PAGE sample buffer was divided into two portions. One was heated at 100 °C for 15 min, and the other was kept at room temperature. Heating in SDS promotes the oligomerization of rhodopsin, so that rhodopsin no longer migrates with an apparent molecular mass of ∼36 kDa in the gel. The heated sample was therefore used to measure protein and radioactivity that was not from rhodopsin in this area (∼36 kDa) of the gel. This background was subtracted from the data obtained from lanes with samples that were not heated. Peripherin/rds, for example, is a photoreceptor outer segment phosphoprotein (29Boesze-Battaglia K. Kong F. Lamba O.P. Stefano F.P. Williams D.S. Biochemistry. 1997; 36: 6835-6846Crossref PubMed Scopus (43) Google Scholar) that has a similar apparent molecular mass. We confirmed by Western blot analysis that bovine peripherin/rds does not oligomerize under the conditions used in the present experiments. Purified ROSs (Method 2, above) from dopamine-treated and control retinas were collected from a sucrose gradient (50 μl), and 10-μl aliquots were suspended in 90 μl of buffer B (10 mm Tris-Cl, pH 7.3, 1 mmEDTA, 1 mm EGTA, 0.05% digitonin). The samples were precipitated with MeOH (90%, 30 min, ice) and centrifuged (20,000 × g, 30 min, 4 °C). Each supernatant was lyophilized and then resuspended in 10 μl of water. A 1-μl aliquot was loaded on to a Cellulose polyethyleneimine TLC plate (8 × 8 cm). Stepwise chromatography was run in the first direction with 0.2 (1 min), 1 (3 min), and 1.6 m LiCl (10 min). The LiCl was removed by washing the plates in MeOH (15 min). Dry plates were used for stepwise chromatography in the second direction with 0.5 (0.5 min), 2 (1 min), and 4 m sodium formate (8 min) (pH 3.4) (30Mangold H. Stahl E. Thin-Layer Chromatography: A Laboratory Handbook. Topan Co., Tokyo1969: 792-802Google Scholar). Radioactivity was quantified using a PhosphorImager. 32P phosphorylation of rhodopsin was performed in intact retinas as above. Rod outer segments from dopamine-treated and control retinas were purified as described (Method 2, above), and their cytosolic fractions (0.6 mg/ml) were used to assay phosphatase activity (1 nmrhodopsin; 22 °C for 30 min, during which time32P release was linear) (7Kutuzov M.A. Bennett N. Eur. J. Biochem. 1996; 238: 613-622Crossref PubMed Scopus (38) Google Scholar, 31Azarian S.M. King A.J. Hallett M.A. Williams D.S. J. Biol. Chem. 1995; 270: 24375-24384Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Radioactive products were separated by SDS-PAGE and analyzed by a PhosphorImager. Paired Student's t tests were performed to determine the probability (p) of no significant difference. After retinas were exposed to a flash of light, rhodopsin was phosphorylated, reaching maximal phosphorylation level after 10 min (27Udovichenko I.P. Newton A.C. Williams D.S. J. Biol. Chem. 1997; 272: 7952-7959Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The rate of phosphorylation in control retinas and in retinas exposed to 100 μm exogenous dopamine was similar (Fig. 1). After 30 min, the level of rhodopsin phosphorylation decreased, with rhodopsin in the dopamine-treated retinas dephosphorylated at a faster rate. By 45 min after the light flash, the level of phosphorylation of rhodopsin in dopamine-treated retinas was only 50% that in control retinas (Fig. 1). A similar result was obtained by three different procedures of sample preparation, as described under “Experimental Procedures”; results from Method 1 are illustrated in Fig. 1. Moreover, this result was found irrespective of whether 80 or 6% of the rhodopsin was photoexcited by the flash (Fig. 2).Figure 2Dopamine has the same effect on the phosphorylation and dephosphorylation of rhodopsin at two levels of flash intensity. Retinas were exposed to a flash of light that excited either 6 or 80% of rhodopsin. The state of rhodopsin phosphorylation was determined from crude ROSs (Method 1). There was no significant difference (NS) between 1 μmdopamine-treated and control samples 15 min after a flash of either intensity. However, 45 min after either flash, rhodopsin in dopamine-treated retinas contained ∼50% less phosphate. Data represent the means ± S.E. and were pooled from six retinas from four different experiments.View Large Image Figure ViewerDownload (PPT) To test whether or not the effect of dopamine resulted in a general effect on ROS protein phosphorylation, we carried out two tests. First, the amount of [32P]ATP was measured in ROSs following incubation of retinas for 45 min in the presence or the absence of dopamine. PhosphorImager analysis of two-dimensional TLC plates showed that in dopamine-treated retinas the amount of ROS [32P]ATP was similar to that in control retinas (102 ± 7%; n = 12; p = 0.94). Second, we observed that the radioactivity of minor phosphoproteins was unaffected by dopamine (Fig. 3). These results are consistent with dopamine having a specific effect on the phosphorylation state of rhodopsin. Further analysis of the reduction in the level of rhodopsin phosphorylation 45 min after the flash showed that it was effected by nanomolar concentrations of exogenous dopamine (Fig. 4). These concentrations are in the range of reported dissociation constants (Kd) for dopamine receptors in the high affinity state (32Seeman P. Van Tol H.H. Trends Pharmacol. Sci. 1994; 15: 264-270Abstract Full Text PDF PubMed Scopus (834) Google Scholar). Dopamine receptors fall into two general classes, D1-like and D2-like. D1 receptors act by activating adenylate cyclase. D2 receptors typically act by inhibiting adenylate cyclase (32Seeman P. Van Tol H.H. Trends Pharmacol. Sci. 1994; 15: 264-270Abstract Full Text PDF PubMed Scopus (834) Google Scholar, 33Sibley D.R. Monsma Jr., F.J. Shen Y. Int. Rev. Neurobiol. 1993; 35: 391-415Crossref PubMed Scopus (215) Google Scholar, 34Ogawa N. Acta Med. Okayama. 1995; 49: 1-11PubMed Google Scholar). To test which class of receptor might be involved in mediating the dopamine effect on rhodopsin phosphorylation, we tested whether antagonists selective for D1-like or D2-like receptors would counter the lowered phosphorylation level found 45 min after the light flash. As illustrated in Fig. 5, SCH-23390, a selective D1 antagonist, did not interfere with the dopamine effect. However, spiperone, a selective D2 antagonist, did; it resulted in a higher level of phosphorylation. This finding is consistent with previous reports identifying D2-like receptors on rod photoreceptors (20Brann M.R. Young III., W.S. Neurosci. Lett. 1986; 69: 221-226Crossref PubMed Scopus (41) Google Scholar, 35Dearry A. Falardeau P. Shores C. Caron M.G. Cell. Mol. Neurobiol. 1991; 11: 437-453Crossref PubMed Scopus (52) Google Scholar, 36Tran V.T. Dickman M. Invest. Ophthalmol. Visual Sci. 1992; 33: 1620-1626PubMed Google Scholar, 37Wagner H.J. Luo B.G. Ariano M.A. Sibley D.R. Stell W.K. J. Comp. Neurol. 1993; 331: 469-481Crossref PubMed Scopus (62) Google Scholar). These results indicate that exposure to dopamine and activation of D2 receptors on photoreceptor cells alters the kinetics of rhodopsin dephosphorylation. One explanation is that dopamine leads to activation of rhodopsin phosphatases. Alternatively, dopamine could lead to preferential phosphorylation at a site that is dephosphorylated more rapidly. Protein kinase C phosphorylates a domain that is not a primary phosphorylation site for rhodopsin kinase (38Greene N.M. Williams D.S. Newton A.C. J. Biol. Chem. 1995; 270: 6710-6717Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 39Greene N.M. Williams D.S. Newton A.C. J. Biol. Chem. 1997; 272: 10341-10344Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and stimulation of protein kinase C phosphorylation of rhodopsin results in faster dephosphorylation (27Udovichenko I.P. Newton A.C. Williams D.S. J. Biol. Chem. 1997; 272: 7952-7959Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). However, altering the relative activities of protein kinase C and rhodopsin kinase results in a different rate of phosphorylation (27Udovichenko I.P. Newton A.C. Williams D.S. J. Biol. Chem. 1997; 272: 7952-7959Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), which was not evident in dopamine-treated samples (Fig. 1). In experiments to test whether ROSs from dopamine-treated retinas contained greater phosphatase activity, purified ROS membranes containing32P-phosphorylated rhodopsin were incubated with ROS cytosol from control or dopamine-treated retinas (7Kutuzov M.A. Bennett N. Eur. J. Biochem. 1996; 238: 613-622Crossref PubMed Scopus (38) Google Scholar, 31Azarian S.M. King A.J. Hallett M.A. Williams D.S. J. Biol. Chem. 1995; 270: 24375-24384Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Fig. 6 illustrates that dopamine-treated ROS cytosol contained significantly more rhodopsin phosphatase activity. Previous work has shown that dopamine may regulate phosphatase-1 via D1 receptors and cyclic AMP-dependent kinase phosphorylation of DARPP-32 (dopamine and cAMP-regulated phosphoprotein), a phosphatase-1 inhibitor (40Hemmings Jr., H.C. Greengard P. Tung H.Y. Cohen P. Nature. 1984; 310: 503-505Crossref PubMed Scopus (479) Google Scholar). However, this is the first report suggesting an effect of dopamine on other phosphatases and on phosphatase activity via D2 receptors. Dephosphorylation of GPCRs has received less attention than their phosphorylation. In comparison to our knowledge of kinases that phosphorylate GPCRs, less is known about the phosphatases that dephosphorylate them, and, in particular, how these phosphatases are regulated. However, dephosphorylation of rhodopsin is necessary to complete the rhodopsin cycle following light activation and then deactivation by phosphorylation and arrestin binding (41Stryer L. Annu. Rev. Neurosci. 1986; 9: 87-119Crossref PubMed Scopus (774) Google Scholar, 42Yarfitz S. Hurley J.B. J. Biol. Chem. 1994; 269: 14329-14332Abstract Full Text PDF PubMed Google Scholar, 43Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 571-576Crossref PubMed Scopus (167) Google Scholar). The importance of rhodopsin dephosphorylation is emphasized byDrosophila rdgC mutants. In the absence of rhodopsin phosphatase (the product of the rdgC gene), the phosphorylation state of rhodopsin is abnormally high, termination of the light response is defective, and the photoreceptor cells degenerate (8Vinos J. Jalink K. Hardy R.W. Britt S.G. Zuker C.S. Science. 1997; 277: 687-690Crossref PubMed Scopus (103) Google Scholar). The present results demonstrate a role for dopamine in the regulation of rhodopsin dephosphorylation and indeed suggest that it effects stimulation of rhodopsin phosphatase. Because dopamine is normally released by cells in the inner retina, these results identify the potential for a novel means of regulation of rhodopsin: from the inner retina back to the light receptor.