Vertebrate photoreceptors are excited by illumination, from which they recover when illumination is reduced and adapt to steady light. Biochemical and electrophysiological studies have revealed an enzymatic cascade involving cyclic GMP that mediates phototransduction. Each step of the cascade is regulated by molecular mechanisms that allow recovery and adaptation. Ca2+ also plays an important role in phototransduction. For example, light-stimulated hydrolysis of cyclic GMP lowers intracellular [Ca2+] which then stimulates phosphorylation of rhodopsin and activation of guanylyl cyclase. The applicant proposes the following three specific aims in order to further understand the molecular aspects of such photoreceptor transduction cascades.
Specific Aim I is to investigate the mechanism by which photoactivated rhodopsin stimulates rhodopsin kinase to phosphorylate other rhodopsins, a process referred to as high gain phosphorylation. The effect of recoverin on high-gain phosphorylation will also be investigated.
Specific Aim II is to identify functional domains of photoreceptor membrane guanylyl cyclases. These studies focus on identifying interactions between the cyclase and a Ca2+-sensitive activator protein. The role of the kinase homology domain within the cyclase and the role of phosphorylation of the cyclase will also be investigated.
Specific Aim III is to identify proteins that are specifically expressed in a particular type of photoreceptor. Differential display polymerase chain reaction will be used with individual rod and cone cells to identify mRNA transcripts that are expressed in one photoreceptor type but not in another. The rationale for this approach is that proteins important to photoreceptor development and function will be identified.
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