INTERACTION OF RGS PROTEINS WITH G BETA SUBUNIT G BETA 5

最近发现的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gtpase激活蛋白（gap）。在体外，这种极高的GAP活性意味着G蛋白传递的信号可以过早终止；因此，研究人员推测RGS蛋白本身的活性应该在体内受到调节。然而，RGS调控的机制目前是已知的。我们最近发现RGS蛋白RGS6、7和9可以在体内和体外与G蛋白β亚基Gbeta5直接相互作用。我们的初步数据表明，Gbeta5可以阻止RGS与Galpha的结合，这表明Gbeta5在主要结构和性质上与其他四个Gbeta亚基有显著不同。与其他总是与gamma亚基相关的Gbeta相比，从天然来源分离的Gbeta5-RGS复合物不含gamma。此外，体外重组Gbeta 5和RGS不需要γ。相反，Gbeta5与RGS分子中的一个结构域结合，该结构域与gamma亚基具有惊人的结构同源性。这种类γ结构域存在于秀丽隐杆线虫的RGS6、RGS7、RGS9和EGL-10中。由于Gbeta5以及RGS的6、7和9主要在中枢神经系统中表达，这种机制似乎是神经元信号传导的特异性机制。本研究将研究Gbeta5-RGS相互作用的功能、特异性和结构基础。AIM1将通过体外重组蛋白的生化分析来描述Gbeta5、RGS和Galpha相互作用时发生的基本分子事件。AIM2将使用免疫学技术确定体内可以发现哪些特定的Gbeta5-RGS- Galpha复合物。它还将表征Galpha， RGS和Gbeta的多种亚型，并使用突变分析确定Gbeta和RGS参与结合的位点。AIM3将致力于了解受Gbeta5-RGS相互作用影响的生理过程，并识别通过该途径发出信号的特定受体。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。此外，这项研究对于开发作为RGS功能调节剂的新疗法具有重要意义。阻断RGS- gbeta5相互作用，例如通过合成模拟结合位点的肽，有望激活RGS并导致特定信号通路的抑制。本研究的基本见解和分析可能有助于设计由该机制起作用的小化合物。

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.