Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase A is a potential mechanism of light- and Ca2+-mediated regulation ofG protein function in photoreceptors

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G p rotein transducin, causing it to bind GTP and stimulate the enzyme cGMP pho sphodiesterase. The rapid termination of the active state of this pathway i s dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Her e, we show that, in preparations of photoreceptor outer segments (OS), RGS9 -1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Prote in kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (P KA) 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 pro tein beta subunit G beta5. Phosphorylated RGS9-1 remains associated with G beta 5L, a photoreceptor-specific splice form, which itself was not phospho rylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by e xogenous PKA. The PKA catalytic subunit could also phosphorylate recombinan t RGS9-1, and mutational analysis localized phosphorylation sites to Ser(42 7) and Ser(428). Substitution of these residues for Glu, to mimic phosphory lation, 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 mecha nisms mediating a stronger photoresponse in dark-adapted cells.