STRUCTURAL AND ENZYMATIC ASPECTS OF RHODOPSIN PHOSPHORYLATION

Photoactivated rhodopsin (Rho) is phosphorylated near the C terminus at multiple sites, predominantly at Ser(334), Ser(338), and Ser(343). We systematically examined the sites of phosphorylation upon flash act ivation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylat ion at Ser(334), Ser(338), and Ser(343), without changing the ration b etween phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (wi th H7), or reconstitution of protein kinase C with purified ROS membra nes. The stoichiometry and the ration between different phosphorylatio n sites in purified Rho were also reproduced using RK, purified to app arent homogeneity form ROS or from an insect cell expression system. T hus, we conclude that light-dependent phosphorylation of Rho is mediat ed primarily by RK. Depalmitoylation of Rho at Cys(322) and Cys(323) a ltered the conformation of the C terminus of Rho, as observed by the p hosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the p resence of four conserved amino acids at the C terminus of Rho. The ac cumulation of phosphorylated Ser(334) observed in vivo could result fr om slower dephosphorylation of this site as compared with dephosphoryl ation of Ser(338) and Ser(343). These data provide a molecular mechani sm for the site-specific phosphorylation of Rho observed in vivo.