CHARACTERIZATION OF NOVEL G-ALPHA INTERACTING PROTEINS

新型 G-α 相互作用蛋白的表征

• 批准号: 6027297
• 负责人: KIM Arthur NEVE
• 金额: $ 7.55万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6027297
• 关键词: DNA binding protein; G protein; gene expression; histochemistry /cytochemistry; immunofluorescence technique; immunologic assay /test; immunoprecipitation; membrane proteins; messenger RNA; protein binding; protein localization; protein protein interaction; protein structure function; yeast two hybrid system

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.

该研究计划的总体目的是鉴定调节异物三聚体G蛋白的α亚基（G-Alpha）活性或亚细胞定位的蛋白质。调节蛋白可能是蛋白质，其与G-α的相互作用以前不被欣赏，或者它们可能是新型蛋白质或蛋白质家族。 G蛋白信号传导（RGS蛋白）的调节剂是最近的例子。 RGS蛋白增强了许多G-Alpha亚基的内在GTPase活性，从而降低G蛋白的活性。在酵母两杂化测定中已经鉴定出与G-Alpha-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AP2的MU亚基。另外两个本质上是未知的，一个与秀丽隐杆线虫基因组项目中鉴定出的未知功能的预测蛋白质同源，另一个仅被描述为编码1405氨基酸蛋白的匿名脑mRNA。在两种杂交测定中NEFA鉴定之后，我们证实了NEFA和G-Alpha-S之间的物理相互作用。本应用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-Alpha-S之间的相互作用。第一个具体目的是由两个杂交测定中蛋白质NEFA，AP2-MU，C16C10.10和AB002373的鉴定的假设驱动的。 1）将确认在初始酵母双杂交筛选中鉴定出的G-Alpha-S和蛋白质之间的物理相互作用。第二个具体目的是基于以下假设：NEFA和G蛋白α亚基之间的物理相互作用在生理上是相关的。 2）将对NEFA及其同源mRNA的亚细胞和区域分布进行表征，并评估NEFA与某些亚型之间的功能相互作用的可能性。第三个具体目的是评估以下假设：在AIM L中确定的给定靶蛋白和G-Alpha-S之间存在物理相互作用，这反映了蛋白质与G-α的某些亚型之间的功能相互作用。 3）将确定G-Alpha与其他三种靶蛋白中的任何一个在AIM L中确认的物理相互作用的功能后果。