Phosphorylation at sites near rhodopsin's carboxyl-terminus regulates light initiated cGMP hydrolysis.

We find prompt, high stoichiometry phosphorylation of rhodopsin in response to low fractional rhodopsin bleaching in bovine rod outer segments (ROS). For example, 4 +/- 1 phosphates are incorporated per bleached rhodopsin (Rho*) in 30 sec and 6 +/- 2 phosphates are incorporated in 75 sec in response to bleaching 1% of the rhodopsin in the presence of 1 mM [gamma-32P]ATP and 0.1 mM nonradioactive GTP. Omission of GTP leads to ca 70% inhibition of rhodopsin phosphorylation, presumably due to the GTP-binding protein blocking access of rhodopsin kinase to rhodopsin. Light induced phosphodiesterase (PDE) activation is rapidly quenched in the presence of ATP as first reported by by Liebman and Pugh [Nature 287, 734-736 (1980)]. The kinetics of rhodopsin phosphorylation vary with conditions and from preparation to preparation, however, they are always at least as fast as the ATP dependent quenching of PDE activation. The maximum extent of rhodopsin phosphorylation was limited by specific proteolytic trimming of the carboxyl-terminal phosphorylation sites in washed ROS membranes "stripped" of extrinsic proteins. Membrane preparations with 6,4,2, or 1 phosphate(s) incorporated/Rho* (after a 75 sec post bleach incubation) were produced by treatment with: no protease, carboxypeptidase Y (C), trypsin (T), or both T and C (TC), respectively, followed by reassociation with extrinsic membrane proteins and phosphorylation. Low fractional bleaching was required for maximum phosphorylation/Rho* in membranes which were stripped and reassociated with extrinsic proteins and in isolated ROS. Removal of C-terminal rhodopsin phosphorylation sites has little or no effect on light activation of PDE in the absence of ATP. However, in the presence of ATP the extent of the removal of C-terminal rhodopsin residues has large effects on the light activation and the shut-off of PDE. The single phosphate/Rho* that was added to TC digested membranes reduced the lifetime of Rho* but apparently was not incorporated rapidly enough under our conditions to inhibit the Vmax of PDE. The two phosphates/Rho* which were incorporated after T digestion cause a large decrease in the lifetime of Rho* as well as a decrease in the Vmax of PDE (at low bleaching levels). The four phosphates/Rho* that were added after C digestion further reduce the lifetime of Rho* and the Vmax of PDE. The 6 phosphates/Rho* which were incorporated into the unproteolyzed membranes have little additional effect on Rho* lifetime compared to 4 phosphates/Rho*. However, increasing the phosphorylation observed from 4 to 6 phosphates greatly inhibits the Vmax of PDE at intermediate bleaching levels.(ABSTRACT TRUNCATED AT 400 WORDS)