The GoLoco motif: a novel Galpha interaction domain

该提案中的具体研究针对FIGS蛋白家族的两个成员，即RGS 12和RGS 14，这两个成员在正常的神经元功能中非常重要。推定的Galphai/o-结合（“GoLoco”）基序的发现使我们假设RGS 12和RGS 14通过修饰GDP/GTP循环或充当Ga亚基的支架来协调异源三聚体G蛋白信号传导途径。我们将确定RGS 12和RGS 14 GoLoco区域的选择性和结构决定因素，通过酵母双杂交，体外共沉淀，表面等离子体共振结合试验和免疫共沉淀研究的组合。我们还将通过体外测量核苷酸结合和水解来确定GoLoco结合对Ga GDP/GTP循环的影响。最后，使用X射线晶体学，我们将确定Ga/GoLoco复合物的原子分辨率结构，以在分子水平上理解GoLoco基序所表现出的特异性和活性。我们的长期目标是确定RGS蛋白活性的分子机制，为与异常G蛋白偶联受体信号传导相关的疾病状态和临床病症提供未来的治疗靶点。本提案中概述的初步目标是定义RGS 12和RGS 14的分离GoLoco区域的结合特异性、结构决定簇和生物化学活性的分子基础，以便更好地理解RGS 12和14在调节神经元信号转导中发挥的作用。

The specific research in this proposal is directed toward two members of the FIGS protein family throught to be important in proper neuronal functioning: RGS12 and RGS14. Discovery of putative Galphai/o- binding ("GoLoco") motifs has led us to the hypothesis that RGS12 and RGS14 coordinate heterotrimeric G-protein signaling pathways by modifying the GDP/GTP cycle or serving as scaffolds for Galpha subunits. We will determine the selectivity and structural determinants of RGS12 and RGS14 GoLoco regions by a combination of yeast-two hybrid, in vitro coprecipitation, surface plasmon resonance binding assays, and co-immunoprecipitation studies. We will also determine the effects of GoLoco association on the Galpha GDP/GTP-cycle through in vitro measurements of nucletide binding and hydrolysis. Finally, using x-ray crystallography, we will ascertain the atomic resolution structure of a Galpha/GoLoco complex to understand, at the molecular level, the specificity and activity demonstrated by GoLoco motifs. Our long term objectives are to define the molecular mechanisms governing RGS protein activity to provide future therapeutic targets for disease states and clinical disorders associated with aberrant Gprotein coupled receptor signaling. The initial objectives outlined in this proposal are to define the molecular basis of the binding specificity, structural determinants, and biochemical activities of isolated GoLoco regions of RGS12 and RGS14 in order to better understand the role that RGS12 and 14 play in regulating signal transduction in neurons.