CRYSTAL STRUCTURES OF PROTEIN AND PROTEIN COMPLEXES INVOLVED IN G PROTEIN SIGNAL

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。 这项提案涉及三个具有共同主题的G蛋白介导的信号传递项目。(1)我们建议确定RGS-Rhogef家族中两个成员的新结构，它们是激活小G蛋白Rho的鸟嘌呤核苷酸交换因子(GEF)。RGS-RhoGEF本身受到G？13的刺激，G？13是一种异源三聚体G蛋白α亚单位。我们将测量与G？13结合的RGS-Rhogef家族中两个成员的复合体的单色数据(如果需要的话，也可以测量SAD或MAD)。RGS-Rhogef家族中的一个成员是G？13的GTP酶激活蛋白(GAP)，另一个不是。我们已经在两个RhoGEF的间隙结构域中产生了嵌合体，并计划确定这些蛋白质的结构以确定GAP活性的基础。(2)哺乳动物膜腺苷酰环化酶(Mac)是由异源三聚体G蛋白α亚单位家族的典型成员G？S激活的。确定了G？S与Mac结合的催化结构域的结构。我们将在G？S不在场的情况下测量催化区域晶体的单色数据，以更好地了解其激活机制。Mac也被二萜阿夫斯柯林激活。我们将确定与forsklin拮抗剂结合的Mac的结构，从而确定潜在的Mac抑制剂。人可溶性腺苷环化酶(SAC)不受G？S调节，而受钙和碳酸氢盐调节。我们将测量SAC催化域晶体的单色衍射数据。(3)Par6是一种细胞分裂调节因子，可同时与两种小G蛋白--CDC42和Rin或Rit结合。Rit和Rin的结构尚不清楚，但我们有GTP和GDP束缚态的晶体，我们将测量其单色数据。制备了与Rit？GDP结合的Par6片段的小晶体，用于单色数据采集。

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. This proposal concerns three projects with a common theme in G protein-mediated signaling. (1)We propose to determine new structures of two members of the RGS-RhoGEF family, which are guanine nucleotide exchange factors (GEFs) that activate the small G protein Rho. RGS-RhoGEFs are themselves stimulated by G?13, a heterotrimeric G protein alpha subunit. We shall measure monochromatic (and if necessary SAD or MAD) data for complexes of two members of the RGS-RhoGEF family bound to G?13. One member of the RGS-RhoGEF family is a GTPase activating protein (GAP) for G?13, and the other is not. We have generated chimeras within the GAP domin of the two RhoGEFs and plan to determine the structures of these proteins to determine the basis of GAP activity. (2) mammalian membrane adenylyl cyclases (mAC)are activated by the G?s, the prototypical member of the family of heterotrimeric G protein alpha subunits. The structure of the catalytic domain of mAC bound to G?s has been determined. We will measure monochromatic data from crystals of the catalytic domain in the absence of G?s to better understand the mechanism of its activation. mAC is also activated by the diterpene forskolin. We shall determine the structure of mAC bound to a forskolin antagonist and therefore a potential mAC inhibitor. Human soluble adenylyl cyclase (sAC) is not regulated by G?s, but rather by calcium and bicarbonate. We will measure monochromatic diffraction data for crystals of the catalytic domain of sAC. (3) Par6 is a regulator of cell division that binds simultaneously to two small G proteins, cdc42 and Rin or Rit. The structures of Rit and Rin are not known, but we have crystals of both in GTP and GDP-bound states for which we shall measure monochromatic data. Small crystals of a Par6 fragment bound to Rit?GDP have been prepared for monochromatic data collection.