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传导范例是通过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蛋白途径还影响单体GTP酶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、粘附和基因转录程序。这些通路的失调经常在癌症和其他疾病中发现。 RhoGEF是Rho蛋白的鸟嘌呤核苷酸交换因子。其中几种直接由异源三聚体G蛋白调节。一个例子是p115-RhoGEF，其可以通过与GEF的RH（RGS同源性）结构域的相互作用由异源三聚体G13蛋白特异性调节。此外，我们已经表明，同源物，PDZRhoGEF，可以结合到其激活的底物，RhoA-GTP，形成一个假定的自激活环。我们建议，以提高我们对这些途径的理解，通过结合详细的研究机制的个别组件和更广泛的研究，以确定频谱和特异性的G蛋白调节，自动调节，和串扰内的一组选择的RhoGEFs，包括Lbc家族。结构研究利用经典晶体学和小角度x射线相结合 通过SAXS散射来鉴定分子之间的调控相互作用的细节。然后可以使用选择性改变功能的特异性诱变来确定调节中的影响。实验集中在使用磷脂囊泡模拟质膜将系统地检查频谱和机制的G蛋白亚基和激活的单体GTP酶的一组靶向RhoGEFs的调节，特别是使用调节本地化的基板和集成的多个输入。体外和i细胞实验将检查Rho和Rac通路之间的交叉调节范例，这可能是细胞形状和迁移协调调节的关键，并测试自动调节机制的生理影响。 完成拟议的实验将增加我们对这些关键途径如何通过识别新的相互作用和增加对途径内和途径间调节机制的了解来调节生长，分化和细胞运动的理解。这将有助于更好地了解各种激素的调节作用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 of 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, adhesion and programs of gene transcription. Misregulation of these pathways is often found in cancer as well as other diseases. RhoGEFs are guanine nucleotide exchange factors for Rho proteins. Several of these are modulated directly by heterotrimeric G proteins. An example is p115-RhoGEF, which can be specifically regulated by the heterotrimeric G13 protein via interaction with the RH (RGS homology) domain of the GEF. In addition, we have shown that a homolog, PDZRhoGEF, can bind to its activated substrate, RhoA-GTP, to form a putative autoactivation loop. We propose to enhance our understanding of these pathways by a combination of detailed studies on the mechanisms of individual components and broader studies to define the spectrum and specificity of G protein regulation, autoregulation, and crosstalk within a select group of RhoGEFs including the Lbc family. Structural studies utilize a combination of classical crystallography and small-angle x-ray scattering (SAXS) to identify details of regulatory interactions between molecules. Specific mutagenesis to selectively alter function can then be used to determine impact in regulation. Experiments focused on the use of phospholipid vesicles to mimic the plasma membrane will systematically examine the spectrum and mechanism of regulation of a group of targeted RhoGEFs by G protein subunits and activated monomeric GTPases, especially the use of regulated localization to substrates and integration of multiple inputs. Experiments in vitro and i cells will examine paradigms of cross-regulation between Rho and Rac pathways that may be key to coordinated regulation in cellular shape and migration and test the physiological impact of autoregulatory mechanisms. Completion of the proposed experiments will increase our understanding of how these key pathways function in the regulation of growth, differentiation and cell motility through identification of new interactions and increased insight into mechanisms of regulation, both within and across pathways. 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.