Signal Transduction Via Receptors and G Proteins

描述（由申请人提供）：调节激素和其他细胞外刺激的主要信号范式是细胞表面受体使用异源三聚体G蛋白。除了直接调节产生第二信使的细胞内酶外，这些受体/G蛋白途径还影响了单体gtpase Ras超家族的几个成员的作用。该家族的成员，如Ras和Rho蛋白，调节细胞生长、分化、形状和粘附。当P115-RhoGEF及其同源物被异源三聚体G13蛋白激活时，可直接调节GTP在RhoA上的交换。反过来，p115-RhoGEF的RGS （G蛋白信号调节因子）结构域可以刺激G13的GTPase活性，从而使其失活。本研究将继续在体外研究这些途径的组成部分之间的调节机制，并试图清楚地定义它们在细胞调节中的生理作用。拟议的研究包括尝试通过使用经典晶体学和小角度x射线散射（SAXS）来确定调节复合物的结构。体外和细胞内偶联反应的突变分析和重构将研究提出的机制，特别是GTPase刺激对促进或激素受体G13 RhoGEF RhoA功能的作用，抑制激素信号传导和细胞膜易位。荧光传感器将被开发并用于评估G13在体内的实际活性和位置，并将其与包括Rho激活、cAMP调节和细胞运动在内的假定功能联系起来。荧光、诱变、外源表达和基因沉默技术被提出来确定信号复合物的作用和信号在这些途径中的特异性，重点是通过受体来调节溶血磷脂酸和鞘氨醇-1-磷酸。已知p115-RhoGEF及其同系物的突变是致癌的，而G13是发育所必需的。进展将增加我们对这些调节生长、分化和细胞运动的关键途径的理解。这将有助于更好地理解各种激素的调节以及这些蛋白质对细胞功能障碍和疾病的贡献。

DESCRIPTION (provided by applicant): A major signaling paradigm for modulation of hormonal and other extracellular stimuli is the use heterotrimeric G proteins by cell surface receptors. Besides direct regulation of intracellular enzymes that produce second messengers, these receptor/G protein pathways influence the action of several members of the Ras superfamily of monomeric GTPases. Members of this family, such as Ras and Rho proteins, regulate cellular growth, differentiation, shape and adhesion. P115-RhoGEF and its homologs can directly modulate the exchange of GTP on RhoA when activated by the heterotrimeric G13 protein. In turn, the RGS (regulator of G protein signaling) domain of p115-RhoGEF can stimulate the GTPase activity of G13 and thus its inactivation. This proposal continues examination of the regulatory mechanisms among components of these pathways in vitro and will attempt to clearly define their physiological roles in cellular regulation. Proposed studies include attempts to determine structures of regulatory complexes by using both classical crystallography and small-angle x-ray scattering (SAXS). Mutational analysis and reconstitution of coupled reactions in vitro and in cells will examine proposed mechanisms, especially the role of GTPase stimulation for either facilitation or Hormone Receptor G13 RhoGEF RhoA Functions inhibition of hormonal signaling and translocation to cellular membranes. Fluorescent sensors will be developed and used to assess the actual activity and location of G13 in vivo and relate this to putative functions including activation of Rho, modulation of cAMP, and cellular motility. Fluorescence, mutagenesis, exogenous expression and gene-silencing techniques are proposed to determine roles for signaling complexes and specificity of signaling in these pathways with a focus on regulation via receptors for lysophosphatidic acid and sphingosine-1-phosphate. Mutations in p115-RhoGEF and its homologues are known to be oncogenic and G13 is required for development. Progress will increase our understanding of these key pathways regulating growth, differentiation and cell motility. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction and disease. PUBLIC HEALTH RELEVANCE: Mutations in p115-RhoGEF and its homologues are known to be oncogenic and the G13 protein is required for development. Progress will increase our understanding of these key pathways in regulating growth, differentiation and cell motility. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction and disease.