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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该提案涉及G蛋白介导的信号传导中具有共同主题的三个项目。 (1)We建议确定RGS-RhoGEF家族的两个成员的新结构，这两个成员是激活小G蛋白Rho的鸟嘌呤核苷酸交换因子（GEF）。 RGS-RhoGEFs本身刺激G？13，异源三聚体G蛋白α亚基。 我们将测量单色（如有必要SAD或MAD）的数据的两个成员的RGS-RhoGEF家庭结合G？13. RGS-RhoGEF家族的成员之一是G？13，另一个不是。 我们已经在两个RhoGEFs的差距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)哺乳动物膜腺苷酸环化酶（mAC）激活的G？s，异源三聚体G蛋白α亚基家族的原型成员。 结合G的mAC的催化结构域的结构？s已确定。 我们将测量单色数据的催化域的晶体中没有G？这是为了更好地了解其激活机制。 mAC也被二萜毛喉素激活。 我们将确定与毛喉素拮抗剂结合的mAC的结构，因此是潜在的mAC抑制剂。人可溶性腺苷酸环化酶（sAC）不受G？s，而是由钙和碳酸氢盐。 我们将测量sAC催化域晶体的单色衍射数据。(3)Par 6是细胞分裂的调节因子，同时与两种小G蛋白cdc 42和Rin或Rit结合。 Rit和Rin的结构尚不清楚，但我们有GTP和GDP结合态的晶体，我们将测量单色数据。 与Rit结合的Par 6碎片的小晶体？国内总产值是为单色数据收集而编制的。

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. 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.