Abstract
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.
Highlights
Ates and deactivates light-activated rhodopsin [5, 6]
Our data reveal that the phosphorylation of rhodopsin by protein kinase C is mechanistically similar to the phosphorylation of other G protein-coupled receptors by second messenger-regulated kinases
The phosphorylation is independent of the liganded state of the receptor, and the primary phosphorylation site, on the carboxyl terminus, differs from that of the G protein-coupled receptor kinase
Summary
Ates and deactivates light-activated rhodopsin [5, 6] In this pathway, absorption of a photon induces isomerization of the receptor's covalently bound ligand, 11-cis retinal, effecting a conformational change that exposes cytoplasmic surfaces on the receptor to allow interaction with transducin [6]. Absorption of a photon induces isomerization of the receptor's covalently bound ligand, 11-cis retinal, effecting a conformational change that exposes cytoplasmic surfaces on the receptor to allow interaction with transducin [6] These exposed surfaces promote binding of rhodopsin kinase and subsequent phosphorylation on the receptor's carboxyl terminus. The sensitivity of rhodopsin phosphorylation to protein kinase C activators in situ and the light-dependent generation of diacylglycerol in rod outer segments support a role for protein kinase C in visual transduction. The phosphorylation is independent of the liganded state of the receptor, and the primary phosphorylation site, on the carboxyl terminus, differs from that of the G protein-coupled receptor kinase
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