Molecular Mechanism of Photoreceptor G Protein Signaling

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DESCRIPTION (provided by applicant): The long-term objective of the proposed program is to investigate molecular mechanisms underlying signaling via the photoreceptor G protein transducin (Gt) in the vertebrate photoreceptor cells. The role and general regulation of transducin during the activation and turnoff phases in visual excitation are well understood. However, our understanding of certain aspects of the function and mechanisms of Gt remains incomplete. Recent studies have confirmed and extended earlier findings of light-dependent translocation of Gt from the outer segments to the inner segments and other compartments of photoreceptor cells. Very little is known about the mechanism of Gtalpha translocation or new potential Gtalpha interacting proteins in the inner segments. Our search for photoreceptor-specific proteins containing G-protein regulatory (GPR)-motifs has revealed that a member of GPR-family, LGN, is present in the inner segments and the synaptic layer of photoreceptor cells, where, as suggested by the preliminary data, it interacts with Gtalpha. The interaction between LGN and Gtalpha, its functional significance, and novel LGN-binding proteins in the retina will be investigated using biochemical approaches in vitro, a mouse model with disrupted LGN/Gtalpha binding, and an LGN knockout mouse model. Another direction of this proposal stems from our previous studies that have indicated the possibility of a novel mechanism of congenital stationary night blindness. Our results suggested a loss of visual signaling in the Nougaret form of night blindness due to the effector defect of the Gtalpha mutant G38D. The molecular mechanism of Nougaret night blindness and the dominant nature of the G38D mutation will be elucidated using a transgenic GtalphaG38D mouse model. The mice will be examined by a combination of immunohistochemical, biochemical, and electrophysiological approaches. Initial characterization of the transgenic mice indicated that the G38D mutant displays deficient light-dependent translocation. This supports the hypothesis that the movement of Gtalpha towards the inner segment is triggered by a specific conformation and/or interactions of transducin. The Gtalpha determinants for the initiation of translocation will be investigated in the RGS9 knockout mice, GTPase-deficient GtalphaQ200L and PDE6-interaction deficient GtalphaI208A transgenic mice. Overall, these studies will help to achieve a better understanding of Gt signaling mechanisms as well as other G protein signaling systems and will provide information relevant to retinal diseases.