The long term goal of the proposed research if to elucidate the role of protein kinase C in rod outer segments of photoreceptors. Specifically, this proposal explores the structure, regulation, and function of a novel protein kinase C activity identified in rod outer segments in the past funding period. Protein kinase C comprises a family of lipid- regulated isozymes that plays a key role in cell regulation. One function of protein kinase C is the phosphorylation and desensitization of receptors to incoming signals. During the past funding period we showed that protein kinase C is a major contributor to the phosphorylation of rhodopsin in intact retinas. We also showed that treatment of retinas with dopamine alters the phosphorylation of rhodopsin in a similar manner as treatment with phorbol esters, potent protein kinase C activators. In other G protein-coupled receptor pathways, second messenger-regulated kinases mediate heterologous desensitization of receptors (the phosphorylation may be initiated by activation of the kinase by a separate receptor) whereas G protein- coupled receptor kinases mediate homologous desensitization (the phosphorylation follows direct stimulation of the receptor). This proposal tests the hypotheses that a structurally distinct isozyme of protein kinase C is present in photoreceptors, and that its function may be to regulate rhodopsin via a heterologous pathway. The importance of this research is that it identifies a novel signaling mechanism in photoreceptors. Three Specific Aims are: I. STRUCTURE of Protein Kinase C in Rod Outer Segments: Molecular Cloning. The goal of this aim is to identify the gene encoding the protein kinase C isozyme expressed in rod outer segments. Biochemical and immunological analyses in the preceding funding period unveiled the presence of a novel member of the Ca2+- sensitive protein kinase Cs in rod outer segments. II. LOCALIZATION and REGULATION of Protein Kinase C in Rod Outer Segments. The goal of this aim is to elucidate where protein kinase C is localized in photoreceptors and how its activity is regulated. As part of this work, we will investigate conditions in intact retinas that promote protein kinase C activation. III. FUNCTION of Protein Kinase C in Rod Outer Segments: Regulation of Rhodopsin. The effect of phosphorylation by protein kinase C on the function of rhodopsin will be explored. Specifically, the effect of this phosphorylation on rhodopsin s macromolecular interactions (transducin coupling, arrestin binding) will be explored.