Molecular Mechanism of Photoreceptor G Protein Signaling

描述(申请人提供)：拟议计划的长期目标是研究脊椎动物感光细胞中通过光感受器G蛋白转导蛋白(GT)进行信号传递的分子机制。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被很好地理解。然而，我们对GT功能和机制的某些方面的了解仍然不完整。最近的研究证实并推广了早期的发现，即光依赖的GT从感光细胞的外节移位到内节和其他隔室。关于Gtalpha易位的机制或新的潜在的Gtalpha内节相互作用蛋白，人们知之甚少。我们对含有G蛋白调节(GPR)基序的光感受器特异蛋白的研究发现，GPR家族的一个成员LGN存在于光感受器细胞的内节和突触层，初步数据表明，它在那里与Gtalpha相互作用。LGN和Gtalpha之间的相互作用，其功能意义，以及视网膜中新的LGN结合蛋白将通过体外生化方法、LGN/Gtalpha结合中断的小鼠模型和LGN基因敲除小鼠模型进行研究。 这一建议的另一个方向源于我们之前的研究，这些研究表明先天性静止性夜盲可能存在一种新的机制。我们的结果表明，由于Gtalpha突变体G38D的效应器缺陷，Nougart形式的夜盲症的视觉信号丢失。使用转基因GtalphaG38D小鼠模型将阐明Nougart夜盲的分子机制和G38D突变的显性本质。这些小鼠将接受免疫组织化学、生化和电生理方法的组合检查。转基因小鼠的初步鉴定表明，G38D突变体表现出缺乏光依赖易位。这支持了Gtalpha向内段移动是由转导蛋白的特定构象和/或相互作用触发的假设。将在RGS9基因敲除小鼠、GTPase缺陷GtalphaQ200L和PDE6相互作用缺陷GtalphaI208A转基因小鼠中研究启动易位的Gtalpha决定因素。总体而言，这些研究将有助于更好地了解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.