Structural Biology of Rho-Mediated Signaling

Rho gtpase介导心血管系统的多种途径，包括平滑肌ca2 +敏化和对血管形成至关重要的细胞迁移现象。该项目的主要目标是确定控制RhoA上游Ca敏化途径的分子机制。这些途径包括一系列核苷酸交换因子（包括PDZRhoGEF和LARG）激活RhoA GTPase，这些核苷酸交换因子包含将这些蛋白与丛蛋白家族的G和受体偶联的结构域，尽管它们直接参与平滑肌生理尚未得到证实。这些全球环境基金的单个结构域以及多结构域片段的结构将通过核磁共振和x射线晶体学以及其他互补的生物物理方法的组合来确定。结合丛蛋白c端PDZ结构域的功能和机制将通过等温滴定量热法和噬菌体展示来确定相互作用的特异性和亲和力。战略目标是了解信号转导膜受体如何最终激活DH-PH结构域串联，从而催化RhoA上的核苷酸交换。后一反应的选择性也将通过定点诱变和X射线晶体学来探测。gef的分离结构域和多结构域片段的功能特性，以及它们的细胞定位，将在PPG内与项目1和3合作研究。

Rho GTPases mediate diverse pathways in the cardiovascular system, including smooth muscle Ca 2+ sensitization and cell migration phenomena critical for the vasculogenesis. The primary objective of the project is to define the molecular mechanisms that control Ca sensitization pathways upstream of RhoA. These pathways involves activation of the RhoA GTPase by a family of nucleotide exchange factors, including PDZRhoGEF and LARG, which contain domains coupling these proteins to G and receptors of the plexin family, although their direct participation in smooth muscle physiology has not been proven. Structures of the individual domains, as well as of the multi-domain fragments of these GEFs will be determined by a combination of NMR and X-ray crystallography, as well as other complementary biophysical methods. The function and mechanism of the PDZ domain which binds the C-terminus of plexins will be assayed by isothermal titration calorimetry and phage display to determine specificity and affinity of interactions. The strategic aim is to understand how signals transduced membrane receptors ultimately activate the DH-PH domain tandem, which catalyzes the nucleotide exchange on RhoA. The selectivity of the latter reaction will also be probed by site-directed mutagenesis and X, ray crystallography. The functional properties of isolated domains and multi-domain fragments of GEFs, as well as their cellular localization, will be studied in collaboration with Project 1 and 3 within this PPG. The project will also focus on the structural biology of plexins and neuropilins, which function upstream of PDZRhoGEF and LARG in many tissues, and which are of primary importance to vasculogenesis. The central domain of plexin is believed to sequester the active form of Rac through a putative CRIB domain, although the boundaries of structural domains have not been determined. Crystallographic and/or NMR studies will determine the molecular architecture of these proteins and the interaction with Rac. Finally, structures of isolated domains of neuropilins, and the regulatory mechanisms of these receptors will also be probed by a synergistic combination of NMR and X-ray crystallography to determine interdomain communication patterns. This work will provide a model for the PDZRhoGEF activation, and will yield data directly relevant to cardiovascular biology.