NOVEL MECHANISM FOR THE ACTIVATION OF RHODOPSIN KINASE - IMPLICATIONSFOR OTHER G-PROTEIN-COUPLED RECEPTOR KINASES (GRKS)

ATP, its nonhydrolyzable analogue, AMP-PNP;and albumin were found to p romote the dissociation of rhodopsin kinase from rod outer segments (R OS) containing photoactivated-rhodopsin (Rho). These features were em bodied in a protocol for the recovery of rhodopsin kinase from incubat ions containing ROS which had been subjected to a wide range of treatm ents. It was found that the supernatants recovered from mixtures conta ining ATP, rhodopsin kinase, and photolyzed ROS membranes catalyzed a Rho-independent peptide phosphorylation as well as dark-phosphorylati on of rhodopsin. The activities of this activated kinase in the two af orementioned assays were 7-8% of the maximum intrinsic activity found in appropriate standard assays (i.e., light-stimulated phosphorylation of rhodopsin and Rho-dependent peptide phosphorylation). The activat ed kinase reverted to its inactive resting-state in a time dependent f ashion, giving a tau(1/2) of decay of similar to 2 min. The intrinsic activity of kinase as measured by the standard assay, however, remaine d constant during this decay period. No positive evidence was found to suggest that the interconversion activated kinase <----> inactive kin ase occurred by a phosphorylation event. Cumulatively, the results sho w that the interaction of rhodopsin kinase ATP complex with Rho leads to the formation, presumably due to the reorganization of the protein structure, of a soluble active kinase species which reverts to the in active resting state in a time-dependent fashion.