The highly invariant single photon response is a salient feature of the photoreceptor. The single photon response is shaped by the kinetics of the amplification cascades as well as the kinetics of deactivation. Although the steps in the amplification pathway are well-elucidated, mechanisms that regulate signal deactivation are still poorly understood. In vitro reconstitution experiments have identified rhodopsin phosphorylation and arrestin binding to be important in rhodopsin inactivation. However, the physiological relevance of these processes in signal inactivation is uncertain because of difficulties in duplicating physiological conditions and to follow real time reaction kinetics in reconstitution experiments. To overcome these limitations, transgenic mouse technology is used to target mutations in the component of the photocascade. Photoreceptors from these transgenic mice then become a resource for single cell recording and biochemical assays. They will use such an approach to investigate mechanisms regulating rhodopsin deactivation. Specifically, they will examine how phosphorylation leads to receptor inactivation in vivo by measuring the kinetics of signal termination in mice expressing a variety of rhodopsin mutants that lack phosphorylation sites at the COOH-terminus. They will dissect and compare the functional roles of arrestin and its COOH-terminal truncated variant by expressing arrestin alone or the truncated isoform alone in the background of arrestin knock-out mice. They will test the hypothesis that in vivo, arrestin binding limits the extent of rhodopsin phosphorylation by rhodopsin kinase and dephosphorylation by the endogenous protein phosphatase 2A. Finally, they will investigate the process of long-term light adaptation (cellular and molecular changes that develop over days to weeks) in the arrestin knock-out mice where constitutive phototransduction is occurring, and to see whether unabated signal flow is a cause for some forms of retinal disorders. The transgenics approach serves to interface biochemistry, electrophysiology and morphology and brings together a multi-disciplinary effort to investigate mechanisms regulating phototransduction. Their findings will also have relevance to other G-protein signaling pathways.
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