Molecular Mechanism of Photoreceptor G Protein Signaling

光感受器G蛋白信号转导的分子机制

• 批准号: 7082044
• 负责人: Nikolai O Artemyev
• 金额: $ 36.01万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082044
• 关键词: G protein; binding proteins; biological signal transduction; chimeric proteins; crosslink; genetically modified animals; laboratory mouse; phosphodiesterases; phosphorylation; posttranslational modifications; protein binding; protein protein interaction; protein structure function; rhodopsin; site directed mutagenesis; transducin; visual photoreceptor; visual phototransduction; G protein; binding proteins; biological signal transduction; chimeric proteins; crosslink; genetically modified animals; laboratory mouse; phosphodiesterases; phosphorylation; posttranslational modifications; protein binding; protein protein interaction; protein structure function; rhodopsin; site directed mutagenesis; transducin; visual photoreceptor; visual phototransduction

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.

描述（由申请人提供）：拟议程序的长期目标是通过脊椎动物光感受器细胞中的光感受器G蛋白透明素（GT）研究基于信号的分子机制。在视觉激发中激活阶段和转交阶段的角色的作用和一般调节已被充分了解。但是，我们对GT功能和机制的某些方面的理解仍然不完整。最近的研究已经证实并扩展了早期的GT从外部段的光依赖性转运到感光细胞的内部段和其他隔室的发现。关于GTALPHA易位或内部细分市场中新的潜在Gtalpha相互作用蛋白的机制知之甚少。我们搜索含有GPR蛋白调节（GPR）-motifs的光感受器特异性蛋白已表明，GPR家庭LGN的成员存在于感光细胞的内部段和突触层中，如初步数据所暗示的那样，它与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型LGN结合蛋白的相互作用将在体外使用生化方法，具有破坏LGN/gtalpha结合的小鼠模型以及LGN敲除小鼠模型。 该提案的另一个方向源于我们以前的研究，这表明先天性静止夜间失明的新机制可能性。我们的结果表明，由于Gtalpha突变体G38D的效应子缺陷，夜眼性的牛糖片形式丧失了视觉信号传导。 Nougaret夜失明的分子机制和G38D突变的主要性质将使用转基因GTALPHAG38D小鼠模型阐明。将通过免疫组织化学，生化和电生理方法的组合来检查小鼠。转基因小鼠的初始表征表明G38D突变体显示出缺乏光依赖性易位。这支持了以下假设：gtalpha向内部段的运动是由特定的构象和/或跨多霉素的相互作用触发的。将在RGS9基因敲除小鼠，GTPase缺陷型GTALPHAQ200L和PDE6相互作用缺陷GTALPHAI208A转基因转基因小鼠中研究易位的GTALPHA决定因素。总体而言，这些研究将有助于更好地了解GT信号传导机制以及其他G蛋白质信号系统，并提供与视网膜疾病有关的信息。