Kinetics and localization of the phosphorylation of rhodopsin by protein kinase C.
Protein kinase C isolated from retina catalyzes the stoichiometric phosphorylation of bovine rhodopsin. Enzymological studies using receptor in rod outer segment membranes stripped of peripheral proteins reveal that the phosphorylation is independent of receptor conformation or liganded state; the half-time for phosphorylation of unbleached (dark-adapted) rhodopsin, bleached (light-activated) rhodopsin, and opsin (chromophore removed) is the same. The phosphorylation by protein kinase C is Ca2+ and lipid regulated; the Km for Ca2+ decreases with increasing concentrations of membrane, consistent with known properties of Ca(2+)-regulated protein kinase Cs. The Km for ATP is 27 microM, with an optimal concentration for MgCl2 of approximately 1 mM. The phosphorylation of rhodopsin by protein kinase C is inhibited by the protein kinase C-selective inhibitor sangivamycin. Proteolysis by Asp-N reveals that all the protein kinase C phosphorylation sites are on the carboxyl terminus of the receptor. Cleavage with trypsin indicates that Ser338, the primary phosphorylation site of rhodopsin kinase, is not phosphorylated significantly; rather, the primary phosphorylation site of protein kinase C is on the membrane proximal half of the carboxyl terminus. The protein kinase C-catalyzed phosphorylation of rhodopsin is analogous to the ligand-independent phosphorylation of other G protein-coupled receptors that is catalyzed by second messenger-regulated kinases.
Greene
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NM
Department of Chemistry, Indiana University, Bloomington 47405.
Williams
D S
DS
Newton
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*
D005136:Eye Proteins; D011741:Pyrimidine Nucleosides; C005699:sangivamycin; D012243:Rhodopsin; D011494:Protein Kinases; D011493:Protein Kinase C; D051606:G-Protein-Coupled Receptor Kinase 1; D008274:Magnesium; D002118:Calcium