Novel mechanism for the activation of rhodopsin kinase: implications for other G protein-coupled receptor kinases (GRK's).
ATP, its nonhydrolyzable analogue, AMP-PNP, and albumin were found to promote the dissociation of rhodopsin kinase from rod outer segments (ROS) containing photoactivated-rhodopsin (Rho*). These features were embodied in a protocol for the recovery of rhodopsin kinase from incubations containing ROS which had been subjected to a wide range of treatments. It was found that the supernatants recovered from mixtures containing ATP, rhodopsin kinase, and photolyzed ROS membranes catalyzed a Rho*-independent peptide phosphorylation as well as dark-phosphorylation of rhodopsin. The activities of this activated kinase in the two aforementioned 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 activated kinase reverted to its inactive resting-state in a time dependent fashion, giving a tau 1/2 of decay of approximately 2 min. The intrinsic activity of kinase as measured by the standard assay, however, remained constant during this decay period. No positive evidence was found to suggest that the interconversion activated kinase <--> inactive kinase occurred by a phosphorylation event. Cumulatively, the results show 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 inactive resting state in a time-dependent fashion.
Dean
K R
KR
Department of Biochemistry, University of Southampton, U.K.
Akhtar
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D003624:Darkness; D004789:Enzyme Activation; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006207:Half-Life; D008027:Light; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology
D005136:Eye Proteins; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D019204:GTP-Binding Proteins