CHARACTERIZATION OF NOVEL G-ALPHA INTERACTING PROTEINS

这项研究计划的总体目标是确定调节异源三聚体G蛋白的阿尔法亚单位(G-α)活性或亚细胞定位的蛋白质。调节蛋白可能是以前未被认识到与G-α相互作用的蛋白质，也可能是新的蛋白质或蛋白质家族。G蛋白信号调节蛋白(RGS蛋白)是最近的一个例子，RGS蛋白增强了许多G-α亚基的固有GTPase活性，从而倾向于降低G蛋白的活性。在酵母双杂交实验中，已经鉴定出四种与G-α-S相互作用的蛋白质。这四种蛋白质中有两种是NEFA和笼状蛋白相关蛋白AP2的MU亚基。另外两个基本上是未知的，一个与线虫基因组计划中确定的功能未知的预测蛋白质同源，另一个仅被描述为编码1405个氨基酸蛋白质的匿名大脑mRNA。在双杂交实验中确定了NEFA之后，我们证实了NEFA与G-α-S之间的物理相互作用。本申请的目的是确定这种相互作用的功能意义，并启动实验来确认和表征其他三种蛋白质与G-α-S之间的相互作用。第一个特定的目的是由这样的假设驱动的，即在双杂交试验中鉴定蛋白质NEFA、AP2-MU、C16C10.10和AB002373是由于这些蛋白质中的每一个都与G-α-S之间的物理作用。1)将证实G-α-S与酵母双杂交初筛中鉴定的蛋白质之间的物理相互作用。第二个具体目的是基于这样的假设，即NEFA和G蛋白α亚基之间的物理相互作用是生理相关的。2)研究NEFA及其同源基因的亚细胞和区域分布，以及NEFA与某些G-α亚型之间功能相互作用的可能性。第三个具体目的是评估假设，即给定的目标蛋白和G-α-S之间存在物理相互作用，如在Aim L中所确定的，反映了蛋白质和G-α的某些亚型之间的功能相互作用。3)将确定G-α与其他三个靶蛋白中的任何一个之间的物理相互作用的功能后果，这在AIM L中得到了证实。

The overall objective of this research program is to identify proteins that regulate the activity or subcellular localization of the alpha subunit (G- alpha) of heterotrimeric G proteins. Regulatory proteins may be proteins whose interaction with G-alpha was not previously appreciated, or they may be novel proteins or protein families. The Regulators of G Protein Signaling (RGS proteins) are a recent example; RGS proteins enhance the intrinsic GTPase activity of many G-alpha subunits, thus tending to decrease the activity of the G proteins. Four proteins that interact with G- alpha-s have been identified in the yeast two-hybrid assay. Two of the four proteins are NEFA and the mu subunit of the clathrin-associated protein AP2. The other two are essentially unknown, one being homologous to a predicted protein of unknown function identified in the C. elegans genome project, and the other described only as an anonymous brain mRNA encoding a 1405 amino acid protein. Subsequent to the identification of NEFA in the two-hybrid assay, we confirmed a physical interaction between NEFA and G-alpha-s. The objective of the present application is to determine the functional significance of this interaction, and to initiate experiments to confirm and characterize the interactions between the other three proteins and G-alpha-s. The first specific aim is driven by the hypothesis that the identification of the proteins NEFA, AP2-mu, C16C10.10, and AB002373 in the two-hybrid assay is due to a physical interaction between each of these proteins and G-alpha-s. 1) The physical interaction between G-alpha-s and proteins identified in the initial yeast two-hybrid screen will be confirmed. The second specific aim is based on the hypothesis that the physical interaction between NEFA and G protein alpha subunits is physiologically relevant. 2) The subcellular and regional distribution of NEFA and its cognate mRNA will be characterized, and the possibility of a functional interaction between NEFA and certain subtypes of G-alpha will be evaluated. The third specific aim is to assess the hypothesis that the existence of a physical interaction between a given target protein and G-alpha-s, as determined in aim l, reflects a functional interaction between the protein and some subtype of G-alpha. 3) Functional consequences of physical interactions, confirmed in aim l, between G-alpha and any of the other three target proteins will be determined.