Phosphorylation of the Regulator of G Protein Signaling RGS9-1 by Protein Kinase A Is a Potential Mechanism of Light- and Ca2+-Mediated Regulation of G Protein Function in Photoreceptors†
In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 remains associated with Gβ5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser427 and Ser428. Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca2+ ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.
Supported by NIH RO1 Grants GM60019 and EY12982 and Fight for Sight Research to Prevent Blindness America Foundation (to V.Z.S.) and by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression (NARSAD) (to K.L.)
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine.
Institut de Pharmacologie Moleculaire et Cellulaire, CNRS.
Address correspondence to this author at the University of Miami School of Medicine, R-189, 1600 NW 10th Ave., Miami, FL 33136. Phone: (305) 243-3430. Fax: (305) 243-4555. E-mail: [email protected] newssun.med.miami.edu.
Neuroscience Program, University of Miami School of Medicine.
This article is cited by 36 publications.
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