Molecular Mechanism of Photoreceptor G Protein Signaling

描述（由申请人提供）：拟议项目的长期目标是研究脊椎动物光感受器细胞中通过光感受器G蛋白转导蛋白（Gt）信号传导的分子机制。在视觉兴奋的激活和关闭阶段，转导蛋白的作用和一般调节是很清楚的。然而，我们对Gt功能和机制的某些方面的理解仍然不完整。最近的研究证实并扩展了早期的发现，即Gt从光感受器细胞的外节段到内节段和其他区室的光依赖易位。关于Gtalpha易位的机制或在内部片段中新的潜在的Gtalpha相互作用蛋白所知甚少。我们对含有g蛋白调控（GPR）基序的光感受器特异性蛋白的研究表明，GPR家族的成员LGN存在于光感受器细胞的内节段和突触层，在那里，根据初步数据，它与Gtalpha相互作用。我们将通过体外生化方法、LGN/Gtalpha结合中断的小鼠模型和LGN敲除小鼠模型来研究LGN和Gtalpha之间的相互作用、其功能意义和视网膜中新的LGN结合蛋白。

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.