MECHANISM OF GALPHA-13 REGULATED RHOGEF ACTIVITY OF P115RHOGEF

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 P115代表了G12类异源三聚体G蛋白和小GTP酶Rho之间的直接联系。P115既是Galpha-13(Ga13)的负性调节因子，也是下游效应因子。在其N端，p115含有一个Rhogef-RGS(RgRGS)结构域，其功能是针对Ga13的GTP酶激活蛋白(GAP)。与Ga13的结合也能刺激其对RhoA的鸟嘌呤核苷酸交换活性。P115的全球环境基金活性是由串联的DH和PH结构域实现的，这两个结构域与N-末端的rgRGS结构域通过150个残基连接区分开。测定了rgRGS结构域与Ga13的络合物和RhoA与RhoA的络合物中的dH-PH结构域。有证据表明，在与Ga13结合时释放的p115内存在抑制机制。也有证据表明，Ga13与p115中的第二个结合位点相互作用，该结合位点位于N-末端rgRGS结构域之外，可能位于DH和PH结构域内。我们认为，Ga13与p115的结合导致了后者的实质性构象变化，可能是在一个或多个铰链点上的结构域旋转。这种构象变化是刺激环境基金p115对RhoA的活性所必需的。我们最近在BioCAT进行了小角X射线散射实验，以确定p115及其与Ga13的络合物的分子包膜。初步数据分析表明，p115或其与Ga13的络合物在溶液中仍为二聚体。目前正在进行详细的建模工作。 4.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. p115RhoGEF (p115) represents a direct link between the G12 class heterotrimeric G proteins and the small GTPase Rho. p115 is both a negative regulator and a downstream effector for Galpha-13 (Ga13). At its N-terminus, p115 possesses a RhoGEF-RGS (rgRGS) domain that functions as a GTPase activating protein (GAP) towards Ga13. Binding to Ga13 also stimulates its Guanine Nucleotide Exchange (GEF) activity towards RhoA. The GEF activity of p115 is carried out by the tandem DH and PH domains, which are separated from the N-terminal rgRGS domain by a 150 residue linker region. Crystal structures of the complex of the rgRGS domain with Ga13, and the DH-PH domains in complex with RhoA have been determined. There is evidence for an inhibitory mechanism within p115 that is released upon binding to Ga13. There is also evidence that Ga13 interacts with a second binding site in p115 that resides outside of the N-terminal rgRGS domain, possibly within the DH and PH domains. We propose that the binding of Ga13 to p115 causes substantial conformational changes in the latter, possibly domain rotation at one or more hinge points. Such conformational changes are required for stimulation of the GEF activity of p115 towards RhoA. We have recently conducted small angle X-ray scattering experiments at BioCAT to determine the molecular envelope of p115, and that of its complex with Ga13. Initial data analysis indicates that p115 or its complex with Ga13 remains a dimer in solution. Detailed modeling is currently underway. 4