Molecular Mechanism of Photoreceptor G Protein Signaling

描述（由申请人提供）：该计划的长期目标是研究脊椎动物感光细胞中感光细胞G蛋白转导素（Gt）信号转导的分子机制。transducin在视觉兴奋的激活和关闭阶段的作用和一般调节是很好理解的。然而，我们对GT的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光依赖性的Gt从感光细胞的外节到内节和其他隔室的易位的早期发现。关于Gtalpha易位的机制或内节中新的潜在Gtalpha相互作用蛋白的机制知之甚少。我们的搜索光感受器特异性蛋白含有G蛋白调节（GPR）基序揭示了GPR家族的成员，LGN，存在于感光细胞的内节和突触层，在那里，如初步数据所示，它与Gtalpha相互作用。LGN和Gtalpha之间的相互作用，其功能的意义，和新的LGN结合蛋白在视网膜将使用生物化学方法在体外，破坏LGN/Gtalpha结合的小鼠模型，和LGN基因敲除小鼠模型进行研究。 这个建议的另一个方向源于我们以前的研究，这些研究表明了先天性静止性夜盲症的新机制的可能性。我们的研究结果表明，由于Gtalpha突变体G38 D的效应缺陷，在Nought形式的夜盲症中视觉信号的丢失。努特夜盲症的分子机制和G38 D突变的显性性质将使用转基因GtalphaG 38 D小鼠模型阐明。将通过免疫组织化学、生物化学和电生理方法的组合对小鼠进行检查。转基因小鼠的初步表征表明，G38 D突变体显示缺乏光依赖性易位。这支持了Gtalpha向内段的运动是由特定构象和/或转导素的相互作用触发的假设。将在RGS 9敲除小鼠、GTP酶缺陷型GtalphaQ 200 L和PDE 6相互作用缺陷型GtalphaI 208 A转基因小鼠中研究转位起始的Gtalpha决定簇。总的来说，这些研究将有助于更好地理解Gt信号机制以及其他G蛋白信号系统，并将提供与视网膜疾病相关的信息。

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.