GROWTH RECEPTOR BINDING PROTEIN 14 RAPH - RAS COMPLEX

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 胰岛素刺激后，胰岛素受体（一种受体酪氨酸激酶）通过几种机制下调，包括通过蛋白酪氨酸磷酸酶（如PTP 1B）去磷酸化和与衔接蛋白Grb 14结合。Grb 14是Grb 7/10/14家族的成员，包括N-末端聚脯氨酸区、Ras-缔合（RA）结构域、普列克底物蛋白同源（PH）结构域、BPS（PH和SH 2之间）区和C-末端Src-同源-2（SH 2）结构域。我们的实验室先前已经表明，Grb 14的BPS区域通过在激酶结构域的活性位点中作为假底物结合而直接抑制胰岛素受体的催化活性。这导致底物磷酸化的抑制，从而下调胰岛素信号传导。C末端SH 2结构域与激酶的磷酸化激活环结合，以增加Grb 14-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，Grb 14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，这两个域的结构耦合。Grb 14的RA和PH结构域被认为分别与小的GT3 Ras和磷酸肌醇相互作用。我们假设Ras激活作为Grb 14负反馈抑制胰岛素信号传导的计时机制。也就是说，Ras与膜磷酸肌醇协调，将Grb 14募集到细胞膜上靠近胰岛素受体，促进BPS和SH 2结构域与激酶结构域的相互作用，导致其抑制。另一个家族成员Grb 10不能很好地与Ras结合，尽管在RA结构域中具有约50%的序列同一性。Grb 14 RA-PH与GTP结合（激活）Ras复合的晶体学研究，我们寻求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Grb 14提供结构基础。

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.