PHOSPHORYLATION OF THE GAMMA-SUBUNIT OF THE RETINAL PHOTORECEPTOR CGMP PHOSPHODIESTERASE BY THE CAMP-DEPENDENT PROTEIN-KINASE AND ITS EFFECT ON THE GAMMA-SUBUNIT INTERACTION WITH OTHER PROTEINS

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is co mposed of P alpha beta and two P gamma subunits. Interaction of P gamm a with P alpha beta or with the or subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photor eceptors. Here we have investigated phosphorylation of P gamma by cAMP -dependent protein kinase and its functional effect on the P gamma int eraction with P alpha beta or T alpha in vitro. P gamma, but not P gam ma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of P-32 radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma i ndicate that only threonine 35 in P gamma is phosphorylated. Phosphory lation of P gamma mutants also reveals that the C and N terminals, of P gamma which are required for the regulation of P alpha beta function s are not involved in the P gamma phosphorylation but that arginine 33 , which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta i nteraction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phospho rylation reduces these inhibitory activities. These observations sugge st that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phos phorylation by cAMP-dependent protein kinase may function for the regu lation of phototransduction in vertebrate rod photoreceptors.