Novel Mechanisms Regulating the Heterotrimeric G Protein Complex

描述（由申请人提供）：激素和神经递质调节细胞生长和行为的各种生理过程。它们的同源细胞表面受体具有7个跨膜（7TM）结构域，通过与G蛋白偶联起作用，促进GTP的解离和随后的装载。当GTP被水解和GTPase活性被具有GTPase加速蛋白活性的G信号传导调节因子（RGS）蛋白加速时，信号传导减弱。最近，我们在拟南芥中发现了一种天然存在的7TM-RGS蛋白（AtRGS1），我们假设它是一种d -葡萄糖受体，具有d -葡萄糖依赖的GAP活性。这是受体gap的第一个例子，也是一类新的d -葡萄糖受体的原型。我们还发现拟南芥G1亚基具有快速的核苷酸交换，使核苷酸水解成为速率限制步骤。这种特性与所有测试的G1亚基的缓慢核苷酸交换特性形成鲜明对比，其中GDP释放是G蛋白循环的限速步骤。因此，我们假设G蛋白周期的调控是在GTP水解步骤，并由AtRGS1介导。最后，我们发现d -葡萄糖代谢物显著提高G1亚基的核苷酸水解速率。显然，拟南芥G蛋白循环包含几个有趣的特性，即不需要GEF的G1亚基的激活，GTP水解步骤的循环调节，以及可能是配体调节的GAP控制循环速率的7TM蛋白。由于拟南芥G1具有人类G1的基本核心结构和功能，因此了解拟南芥G1激活的调控机制将为控制人类G1激活提供新的机制。这里的目标是了解G1蛋白是如何在糖信号的背景下被激活的。假设驱动和发现驱动两种方法都将被用来精确地确定什么样的结构赋予监管控制。我们对拟南芥G蛋白周期的初步研究说明了G蛋白周期如何通过经典GEF以外的机制进行调节。因此，周期的更大程度的可塑性现在得到赞赏，并揭示了新的监管切入点。了解这些新机制背后的结构将为调节其他G蛋白周期提供新的手段。在人类中，7TM受体和RGS蛋白通过接头蛋白直接或间接相互作用；这是提供受体和RGS蛋白之间选择性的几种可能机制中的两种。AtRGS1是提供受体- rgs蛋白选择性机制的最极端的例子，因为GEF和GAP都是一个分子上的两个结构域。了解AtRGS1如何以配体依赖的方式调节G蛋白周期，为通过药物治疗调节G蛋白周期开辟了新的可能性。最后，使用拟南芥作为模型将使我们能够解决细胞如何在多细胞生物的背景下对d -葡萄糖作出反应。拟议工作的基本原理是，对不同信号通路中使用的分子机制的理解将产生新的药物靶点，操纵人类信号通路的新思路，以及设计人类信号通路的新工具。

DESCRIPTION (provided by applicant): Hormones and neurotransmitters modulate a variety of physiological processes in cell growth and behavior. Their cognate cell surface receptors, which have seven transmembrane (7TM) domains, act by coupling to G proteins, promoting the dissociation of GDP and the subsequent loading of GTP. Signaling abates when GTP is hydrolyzed and GTPase activity is accelerated by Regulators of G Signaling (RGS) proteins having GTPase accelerating protein (GAP) activity. Recently, we discovered a naturally-occurring 7TM-RGS protein in Arabidopsis (AtRGS1) that we hypothesize to be a D-glucose receptor that has a D-glucose-dependent GAP activity. It is the first example of a receptor-GAP and is the prototype for a new class of D-glucose receptors. We also showed that the Arabidopsis G1 subunit has rapid nucleotide exchange making nucleotide hydrolysis the rate limiting step. This property is in marked contrast to the slow nucleotide exchange property of all tested G1 subunits where GDP release is the rate limiting step of the G protein cycle. Thus, we hypothesize that regulation of the G protein cycle is at the GTP hydrolysis step and is mediated by AtRGS1. Finally, we showed that a D-glucose metabolite dramatically increases the nucleotide hydrolysis rate of the G1 subunit. Clearly, the Arabidopsis G protein cycle contains several interesting properties, namely activation of a G1 subunit that does not require a GEF, regulation of the cycle at the GTP hydrolysis step, and a 7TM protein that may be the ligand-regulated GAP controlling the cycling rate. Because the Arabidopsis G1 has the basic core structure and function of human G1, an understanding of how the Arabidopsis G1 activation is regulated will provide insight into novel mechanisms to control human G1 activation. The goal here is to understand how the G1 protein is activated in the context of sugar signaling. Both hypothesis- and discovery-driven approaches will be taken to determine precisely what structure imparts regulatory control. Our initial study of the Arabidopsis G protein cycle illustrated how the G-protein cycle can be regulated by mechanisms apart by the classical GEF. Consequently, a greater degree of plasticity of the cycle is now appreciated and new entry points for regulation are revealed. Understanding the structure underlying these new mechanisms will provide a new means to regulate other G protein cycles. In humans, 7TM receptors and RGS proteins interact either directly or indirectly via adaptor proteins; these are two of several possible mechanisms providing selectivity between receptors and RGS proteins. AtRGS1 is the most extreme example of a mechanism providing receptor-RGS protein selectivity in that both GEF and GAP are two domains on one molecule. Understanding how AtRGS1 regulates the G protein cycle in a ligand dependent manner opens up new possibilities to regulate G protein cycles through drug therapies. Finally, use of Arabidopsis as a model will enable us to solve how cells respond to D-glucose within the context of a multicellular organism. PUBLIC HEALTH RELEVANCE The rationale for the proposed work is that an understanding of molecular mechanisms used in divergent signaling pathways will yield new drug targets, new ideas for manipulating human signaling pathways, and new tools to engineer human pathways.