Signal Transduction Via Receptors and G Proteins

摘要 调节激素和其他细胞外刺激的一个主要信号范式是使用 异三聚体G蛋白通过细胞表面受体。除了对细胞内酶的直接调节外， 产生第二信使，这些受体/G蛋白通路影响几个成员的行动 RAS超家族单体GTP酶。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。P115-Rhogef及其同系物可直接调节 当被异三聚体G13蛋白激活时，GTP在RhoA上的交换。反过来，RGS(监管机构) P115-Rhogef的G蛋白信号转导结构域可以刺激G13的GTPase活性，从而使其失活。 该提案继续审查这些途径各组成部分之间的调控机制 并将尝试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)。体外和体内偶联反应的突变分析和重组 细胞将研究已提出的机制，特别是GTP酶刺激对促进或 激素受体G13 Rhogef RhoA的功能 抑制激素信号和向细胞膜的转位。荧光传感器将是 开发并用于评估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Rho的激活、cAMP的调节和细胞运动。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和 这些通路中信号的特异性，重点是通过溶血磷脂酸和 鞘氨醇-1-磷酸。 已知p115-Rhogef及其同系物的突变是致癌的，G13是必需的 发展。进展将增加我们对这些调控增长的关键途径的理解， 分化和细胞运动。这将有助于更好地理解各种不同的 荷尔蒙和这些蛋白质对细胞功能障碍和疾病的贡献。

Summary 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 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 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.