GTP BINDING PROTEINS AND INSULIN RECEPTOR SINGNALING

GTP 结合蛋白和胰岛素受体信号传导

• 批准号: 2152112
• 负责人: CHIN K SUNG
• 金额: $ 10.86万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2152112
• 关键词: G protein; HTC cell; antibody; biological signal transduction; chemical association; chimeric proteins; guanine nucleotide binding protein; immunoprecipitation; insulin receptor; protein structure function; receptor expression; transfection; western blottings

DESCRIPTION: (Adapted from the applicant's abstract): The objective of the proposed research is to study the intracellular signaling mechanisms for the insulin receptor (IR). The investigators have recently made the observation that after insulin stimulation of rat HTC hepatoma and other cells one or more signaling complexes form that consists of: the IR; the p85 subunit of phosphatidylinositol-3-kinase; Ras GTPase activating protein (GAP); and p62 GAP-associated protein. In the proposal, it is proposed to test the hypothesis that p85, via its two SH2 and one SH3 domains, serves as an adaptor molecule to directly link the activated IR to GTP binding proteins. First, the investigators will study the Ras pathway. Using cell lysates following insulin treatment, it is proposed to study the cellular proteins (IR and p62) interacting with the SH2 domains of p85. For this study, the investigators will employ fusion proteins containing the SH2 and SH3 domains of p85 incorporated into maltose binding protein. Cellular proteins interacting with the fusion proteins will be analyzed by western blotting with appropriate antibodies. To study direct protein-protein interactions, the investigators will employ purified IR and p62; and fusion proteins containing SH2/3 domains of p85 or GAP. To study the role of GAP and p62 in Ras activation, the investigators will employ purified p62 and antibodies to p62 in permeabilized cells and determine the effects on GAP and Ras activities. Second, PI will study the heterotrimeric G protein. The investigators will identify the 41 kD G protein using antibodies to various G proteins. The investigators will also determine whether the 41 kD G protein that they find associated with p85 is also associated with the IR; and is related to the G protein (G1R41) that is associated with the IR in human placenta. To determine whether the 41 kD G protein is associated with regulatory proteins of the Ras pathway, the investigators will immunoprecipitate the 41 kD G protein with specific antibodies and analyze by western blotting with appropriate antibodies. In cells overexpressing normal Ras or dominant inhibitory Ras, the investigators will also study association of this G protein with both p85 and the IR. These proposed studies therefore should provide new insights into IR signaling mechanisms in target cells.

描述：（改编自申请人的摘要）： 拟议的研究是研究细胞内信号传导机制 对于胰岛素受体（IR）。 调查人员最近使 观察到胰岛素刺激大鼠HTC肝癌和其他 细胞一个或多个信号传导复合物由：IR组成； 磷脂酰肌醇-3-激酶的p85亚基； RAS GTPase激活 蛋白质（间隙）；和p62间隙相关蛋白。 在提案中，是 提议通过其两个SH2和一个SH3检验p85的假设 域，充当适配器分子，直接连接激活的 IR与GTP结合蛋白。 首先，调查人员将研究RAS 路径。 在胰岛素治疗后使用细胞裂解物，提出 研究与SH2相互作用的细胞蛋白（IR和p62） p85的域。 对于这项研究，研究人员将采用融合 包含p85的SH2和SH3结构域的蛋白质融合到 麦芽糖结合蛋白。 细胞蛋白与融合相互作用 蛋白质将通过适当的蛋白质印迹分析 抗体。 为了研究直接蛋白质 - 蛋白质相互作用， 调查人员将使用纯化的IR和P62；和融合蛋白 包含p85或间隙的SH2/3域。 研究差距和 P62在RAS激活中，研究人员将采用纯化的P62和 透明细胞中对p62的抗体，并确定对 差距和RAS活动。 第二，PI将研究异三聚体G 蛋白质。 研究人员将使用使用的41 kD G蛋白使用 各种G蛋白的抗体。 调查人员还将确定 他们发现与p85相关的41 kD G蛋白是否也是 与IR相关；并且与G蛋白（G1R41）有关 与人类胎盘中的IR相关。 确定41是否 KD G蛋白与RAS途径的调节蛋白有关， 研究人员将与41 kD G蛋白免疫沉淀 特定的抗体和通过蛋白质印迹分析 抗体。 在过表达正常RA或显性抑制的细胞中 RAS，研究人员还将研究该G蛋白的关联 与P85和IR一起使用。 因此，这些提出的研究应该 对目标细胞中的IR信号传导机制提供新的见解。