GROWTH RECEPTOR BINDING PROTEIN 14 RAPH - RAS COMPLEX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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)被几种机制下调，包括被蛋白酪氨酸磷酸酶(如PTP1B)去磷酸化和结合适配蛋白Grb14。Grb14是Grb7/10/14家族的成员，它包括一个N-末端多聚脯氨酸区、一个RAS结合(RA)结构域、一个Pleckstrin-Homology(PH)结构域、一个BPS(在PH和SH2之间)和一个C-末端Src-Homology-2(SH2)结构域。我们的实验室先前已经证明，Grb14的BPS区通过作为假底物结合在激活域的活性部位而直接抑制胰岛素受体的催化活性。这会抑制底物的磷酸化，从而下调胰岛素信号。我们实验室的生化研究表明，Grb14介导的胰岛素信号的抑制也需要有功能的RA和PH结构域。我们的结晶学研究表明，这两个结构域在结构上是耦合的。Grb14的RA和PH结构域被认为分别与小GTP酶RAS和磷脂酰肌醇相互作用。我们假设RAS的激活是Grb14负反馈抑制胰岛素信号的计时机制。也就是说，RAS与膜磷酸肌醇结合，在靠近胰岛素受体的细胞膜上募集Grb14，促进BPS和SH2结构域与激活域的相互作用，导致其抑制。我们有生化数据表明，与RAS的相互作用是Grb14和Grb7特有的。另一个家族成员Grb10与RAS结合不好，尽管在RA结构域有约50%的序列同源性。通过对Grb14 RA-PH与GTP结合的(活化的)RAS的结晶学研究，我们寻求邮件同步加速器数据收集，将有助于揭示这两种蛋白之间的结合方式，并为激活的RAS招募Grb14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.