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 Ca and lipid regulated; the K for Ca decreases with increasing concentrations of membrane, consistent with known properties of Ca-regulated protein kinase Cs. The K for ATP is 27 µM, with an optimal concentration for MgCl 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 Ser, 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.

FOOTNOTES

*

This work was supported by National Eye Institute Grant EY08820, by a grant from the Fight For Sight Research Division of the National Society to Prevent Blindness (to N. M. G.), and a National Science Foundation Young Investigator award (to A. C. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L39909[GenBank].

§

Present address: Dept. of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0640.

¶

To whom correspondence should be addressed. Tel.: 619-534-4527; Fax: 619-534-6833.

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The abbreviations used are: PMA, phorbol myristate acetate; BAEE, N-benzoyl-L-arginine ethyl ester; DTT, dithiothreitol; MOPS, 3-[N-morpholino]propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

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As an example of high concentrations of one activator reducing the regulation by Ca, rhodopsin reconstituted in membranes containing 95 mol % phosphatidylserine and 5 mol % diacylglycerol is phosphorylated by protein kinase C in the absence of Ca(11) .

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L. M. Keranen and A. C. Newton, unpublished data.

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