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 beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, 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 Ser(427) and Ser(428). 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 Ca(2+) 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.
Balasubramanian
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N
Department of Molecular and Cellular Pharmacology and Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
Levay
K
K
Keren-Raifman
T
T
Faurobert
E
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Slepak
V Z
VZ
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000502:physiology; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014785:Vision, Ocular / Q000235:genetics / Q000502:physiology*
D002614:Chelating Agents; D020690:GTPase-Activating Proteins; D020710:RGS Proteins; C116925:regulator of g-protein signaling 9; D012694:Serine; D004533:Egtazic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium