GROWTH RECEPTOR BINDING PROTEIN 14 RAPH - RAS COMPLEX

生长受体结合蛋白 14 RAPH - RAS 复合物

• 批准号: 8363348
• 负责人: STEVAN R. HUBBARD
• 金额: $ 0.19万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363348
• 关键词: Active Sites; Affinity; Binding; Binding Proteins; Biochemical; C-terminal; Cell membrane; Complex; Coupled; Crystallography; Data; Data Collection; Down-Regulation; Family; Family member; Feedback; Funding; GTP Binding; Grant; Growth; Insulin; Insulin Receptor; Laboratories; Light; Macromolecular Complexes; Mails; Mediating; Membrane; Monomeric GTP-Binding Proteins; N-terminal; National Center for Research Resources; PH Domain; PTPN1 gene; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Principal Investigator; Proline; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Research; Research Infrastructure; Resources; Source; Specificity; Synchrotrons; Time; United States National Institutes of Health; adapter protein; base; cost; insulin signaling; member; platelet protein P47; receptor binding

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. After insulin stimulation, the insulin receptor, a receptor tyrosine kinase, is downregulated by several mechanisms, including dephosphorylation by protein tyrosine phosphatases such as PTP1B and binding of the adapter protein Grb14. Grb14, a member of the Grb7/10/14 family, comprises an N-terminal poly-proline region, a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. Our laboratory has previously shown that the BPS region of Grb14 directly inhibits the catalytic activity of the insulin receptor by binding as a pseudo-substrate in the active site of the kinase domain. This results in suppression of substrate phosphorylation and hence downregulation of insulin signaling. The C-terminal SH2 domain binds to the phosphorylated activation loop of the kinase to increase the affinity and specificity of the Grb14-insulin receptor interaction.Biochemical studies in our laboratory have shown that Grb14-mediated inhibition of insulin signaling also requires functional RA and PH domains. Our crystallographic studies showed that the two domains are structurally coupled. The RA and PH domains of Grb14 are thought to interact with the small GTPase Ras and phosphoinositides, respectively. We hypothesize that Ras activation serves as a timing mechanism for the negative-feedback inhibition of insulin signaling by Grb14. That is, Ras, in coordination with membrane phosphoinositides, recruits Grb14 to the cell membrane in close proximity to the insulin receptor, facilitating the interaction of BPS and SH2 domains with the kinase domain, resulting in its inhibition.The interaction with Ras, for which we have biochemical data, is specific to Grb14 and Grb7. The other family member, Grb10, does not bind well to Ras despite ~50% sequence identity in the RA domain. Crystallographic studies of Grb14 RA-PH in complex with GTP-bound (activated) Ras, for which we seek mail-in synchrotron data collection, will shed light on the mode of binding between these two proteins and provide the structural basis for the recruitment of Grb14 by activated Ras.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 胰岛素刺激后，胰岛素受体（一种受体酪氨酸激酶）通过多种机制下调，包括蛋白酪氨酸磷酸酶（如 PTP1B）的去磷酸化和接头蛋白 Grb14 的结合。 Grb14 是 Grb7/10/14 家族的成员，包含 N 端聚脯氨酸区域、Ras 相关 (RA) 结构域、普莱克斯特林同源 (PH) 结构域、BPS（PH 和 SH2 之间）区域和 C 端 Src-同源-2 (SH2) 结构域。我们实验室此前已表明，Grb14的BPS区域通过作为假底物结合在激酶结构域的活性位点，直接抑制胰岛素受体的催化活性。这导致底物磷酸化受到抑制，从而下调胰岛素信号传导。 C 端 SH2 结构域与激酶的磷酸化激活环结合，增加 Grb14-胰岛素受体相互作用的亲和力和特异性。我们实验室的生化研究表明，Grb14 介导的胰岛素信号传导抑制也需要功能性 RA 和 PH 结构域。我们的晶体学研究表明这两个域在结构上是耦合的。 Grb14 的 RA 和 PH 结构域被认为分别与小 GTPase Ras 和磷酸肌醇相互作用。我们假设 Ras 激活是 Grb14 负反馈抑制胰岛素信号传导的定时机制。也就是说，Ras 与膜磷酸肌醇协同，将 Grb14 招募到细胞膜上靠近胰岛素受体的位置，促进 BPS 和 SH2 结构域与激酶结构域的相互作用，从而抑制激酶结构域。我们有生化数据，与 Ras 的相互作用是 Grb14 和 Grb7 所特有的。另一个家族成员 Grb10 与 Ras 的结合效果不佳，尽管 RA 结构域的序列同一性约为 50%。 Grb14 RA-PH 与 GTP 结合（激活）Ras 复合物的晶体学研究，为此我们寻求邮寄同步加速器数据收集，将揭示这两种蛋白质之间的结合模式，并为激活 Ras 招募 Grb14 提供结构基础。