CHARACTERIZATION OF NOVEL G-ALPHA INTERACTING PROTEINS

本研究计划的总体目标是鉴定调节异源三聚体G蛋白α亚基（G-α）活性或亚细胞定位的蛋白质。调节蛋白可以是以前未认识到与G-α相互作用的蛋白，或者它们可以是新的蛋白或蛋白家族。G蛋白信号调节蛋白（Regulators of G Protein Signaling，RGS蛋白）是最近的一个例子; RGS蛋白增强许多G-α亚基的内在GT3活性，因此倾向于降低G蛋白的活性。已经在酵母双杂交测定中鉴定了与G-α-s相互作用的四种蛋白质。四种蛋白质中的两种是NEFA和网格蛋白相关蛋白AP 2的mu亚基。另外两个基本上是未知的，一个与C. elegans基因组计划，而另一个仅描述为编码1405个氨基酸的蛋白质的匿名脑mRNA。在双杂交试验中鉴定NEFA之后，我们证实了NEFA和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，并启动实验以确认和表征其他三种蛋白质与G-α-s之间的相互作用。第一个具体目标是由以下假设驱动的：在双杂交测定中对蛋白NEFA、AP 2-mu、C16C10.10和AB 002373的鉴定是由于这些蛋白中的每一个与G-α-s之间的物理相互作用。1)将确认G-α-s与初始酵母双杂交筛选中鉴定的蛋白质之间的物理相互作用。第二个具体目标是基于NEFA和G蛋白α亚基之间的物理相互作用是生理相关的假设。2)NEFA及其同源mRNA的亚细胞和区域分布将被表征，NEFA和某些G-α亚型之间的功能性相互作用的可能性将被评估。第三个具体目标是评估这样的假设：如目标1所确定的，给定靶蛋白与G-α-s之间存在物理相互作用，反映了该蛋白与G-α的某些亚型之间的功能相互作用。3)将确定在目标1中确认的G-α与其他三种靶蛋白中的任何一种之间的物理相互作用的功能后果。

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.