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蛋白终止信号转导过程中起着关键作用。RGS是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被提前终止；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的调节机制目前是已知的。这一建议是基于我们最近的发现，即RGS蛋白RGS6、7和9在体内和体外都可以直接与G蛋白β亚基Gbeta5相互作用。我们的初步数据表明，Gbeta5阻止了RGS与Galpha的结合，这表明Gbeta5在一级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GGamma亚单位相关的Gbeta不同，从自然来源分离的Gbeta5-RGS复合体不含有GGamma。此外，重组Gbeta5和RGS的体外重组不需要GGamma。相反，Gbeta5结合到RGS分子中的一个结构域，该结构域与GGamma亚基具有惊人的结构同源性。这个GGamma样结构域存在于RGS6、RGS7、RGS9和EGL-10中，EGL-10是线虫的一种RGS。由于Gbeta5以及RGS、6、7和9主要在中枢神经系统表达，这一机制似乎是神经元信号传递的特异性机制。拟议的研究将研究Gbeta5-RGS相互作用的功能、其特异性和结构基础。AIM1将利用体外重组的蛋白质的生化分析来描述Gbeta5、RGS和Galpha之间相互作用时发生的基本分子事件。AIM2将利用免疫学技术确定在体内可以发现哪些特定的Gbeta5-RGS-Galpha复合体。它还将描述Galpha、RGS和Gbeta的多种异构体，并使用突变分析确定参与结合的Gbeta和Rgs的位置。AIM3将寻求对Gbeta5-RGS相互作用影响的生理过程的理解，以及通过这一途径发出信号的特定受体的识别。这些实验将帮助我们了解RGS蛋白是如何控制神经元中的信号的。此外，本研究对于开发作为RGS功能调节剂的新疗法具有重要意义。例如，通过模拟结合位点的合成肽来阻断RGS-Gbeta5的相互作用，预计会激活RGS，并导致特定信号通路的抑制。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分子化合物。

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.