Signal Transduction Via Receptors and G Proteins

描述(申请人提供)：调节激素和其他细胞外刺激的一个主要信号范例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蛋白通路还影响单体GTP酶RAS超家族的几个成员的活动。这个家族的成员，如Ras和Rho蛋白，调节细胞的生长、分化、形状和黏附。当被异三聚体G13蛋白激活时，P115-Rhogef及其同系物可以直接调节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。这项提案继续在体外检查这些通路的组成部分之间的调节机制，并将试图清楚地定义它们在细胞调节中的生理作用。建议的研究包括尝试使用经典结晶学和小角X射线散射(SAXS)来确定调控络合物的结构。体外和细胞内偶联反应的突变分析和重组将检验所提出的机制，特别是GTPase刺激对G13或激素受体G13 Rhogef RhoA功能的促进作用，抑制激素信号和向细胞膜的转位。将开发和使用荧光传感器来评估G13在体内的实际活性和位置，并将其与可能的功能联系起来，包括激活Rho，调节cAMP和细胞运动。荧光、诱变、外源表达和基因沉默技术被用来确定信号复合体在这些通路中的作用和信号的特异性，重点是通过溶血磷脂酸和鞘氨醇-1-磷酸的受体进行调节。已知p115-Rhogef及其同系物的突变是致癌的，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所赋予的调节以及这些蛋白质对细胞功能障碍和疾病的贡献。 公共卫生相关性：已知p115-Rhogef及其同系物的突变是致癌的，G13蛋白是发育所必需的。进展将增加我们对这些调控生长、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所赋予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.