INTERACTION OF RGS PROTEINS WITH G BETA SUBUNIT G BETA 5

最近鉴定的RGS蛋白家族在通过异源三聚体G蛋白终止信号转导中起关键作用。RGS作为G蛋白α亚基的GTP酶激活蛋白（GAP）。这种体外极高的GAP活性意味着G蛋白传递的信号可以过早终止;因此，研究人员假设RGS蛋白本身的活性应该在体内受到调节。然而，RGS调节的机制目前是已知的。该提议基于我们最近的发现，即RGS蛋白RGS 6、7和9可以在体内和体外与G蛋白β亚基Gbeta 5直接相互作用。我们的初步数据表明，Gbeta 5阻止RGS与Ga的结合，表明Gbeta 5在其一级结构和性质上与其他四种Gbeta 5亚基显著不同。与总是与G γ亚基相关的其他G β相反，从天然来源分离的G β 5-RGS复合物不含G γ。此外，体外重组G β 5和RGS的重建不需要G γ。相反，G β 5结合到RGS分子中的结构域，该结构域与G γ亚基具有显著的结构同源性。该Ggamma样结构域存在于RGS 6、RGS 7、RGS 9和EGL-10中，EGL-10是来自C.优雅由于G β 5以及RGS 6、7和9主要在CNS中表达，因此该机制似乎对神经元中的信号传导具有特异性。该研究将研究Gbeta 5-RGS相互作用的功能，其特异性和结构基础。AIM 1将使用体外重构蛋白的生化分析来描绘在G β 5、RGS和G α之间相互作用时发生的基本分子事件。AIM 2将使用免疫学技术确定可以在体内发现哪些特定的G β 5-RGS-G α复合物。它还将表征G α、RGS和G β的多种亚型，并使用突变分析确定参与结合的G β和RGS的位点。AIM 3将追求通过Gbeta 5-RGS相互作用的生理过程的理解，并通过这一途径信号的特定受体的识别。这些实验将帮助我们了解RGS蛋白如何控制神经元中的信号传导。此外，这项研究对于开发作为RGS功能调节剂的新疗法非常重要。阻断RGS-G β 5相互作用，例如通过模拟结合位点的合成肽，预期会激活RGS并导致特异性信号传导回路的抑制。这项研究开发的基本见解和分析方法可能有助于设计通过这种机制发挥作用的小化合物。

The recently identified family of RGS proteins plays a key role in the termination of signal transduction through the heterotrimeric G proteins. RGS act as GTPase-activating proteins (GAPs) for the G protein alpha subunits. This extremely high GAP activity in vitro implies that the signaling transmitted by G proteins can be terminated prematurely; therefore, researchers postulated that the activity of RGS proteins themselves should be regulated in vivo. However, the mechanisms of RGS regulation are currently now known. This proposal is based on our recent discovery that RGS proteins RGS6, 7 and 9 can directly interaction, in vivo and in vitro, with the G protein beta subunit Gbeta5. Our preliminary data demonstrate that Gbeta5 prevents the binding of RGS to Galpha, indicating that Gbeta5 which is significantly different from the four other Gbeta subunits in its primary structure and properties. In contrast to other Gbeta's which are always associated with a Ggamma subunit, the Gbeta5-RGS complexes isolated from native sources do not contain Ggamma. Additionally,, Ggamma is not required for the reconstitution of recombinant Gbeta 5 and RGS in vitro. Instead, Gbeta5 binds to a domain in the RGS molecule which has a striking structural homology to the Ggamma subunits. This Ggamma-like domain is present in RGS6, RGS7, RGS9 and EGL-10, an RGS from C. elegans. Since Gbeta5 as well as the RGS's, 6, 7 and 9 are predominantly expressed in the CNS, this mechanism appears to be specific for signaling in neurons. The proposed research will study the function of the Gbeta5-RGS interaction, its specificity and structural basis. AIM1 will delineate the basic molecular events that occur upon the interaction between Gbeta5, RGS and Galpha using the biochemical analysis of the proteins reconstituted in vitro. AIM2 will determine which particular Gbeta5-RGS- Galpha complexes can be found in vivo using immunological techniques. It will also characterize the multiple isoforms of Galpha, RGS and Gbeta and determine the sites of Gbeta and RGS involved in the binding using mutational analysis. AIM3 will pursue the understanding of the physiologic processed affected by Gbeta5-RGS interaction and the identification of specific receptors that signal through this pathway. These experiments will help us understand how RGS proteins control the signaling in neurons. In addition, this research is important for the development of novel therapeutics which act as regulators of RGS function. Blocking the RGS-Gbeta5 interaction, for instance by synthetic peptide that mimics the binding sites, is expected to activate the RGS and lead to inhibition of a specific signaling circuit. The basic insights and the assays developed by this research might help to design small compounds acting by this mechanism.