IDENTIFICATION OF PKD1 PROTEIN BINDING PARTNERS

PKD1 蛋白结合伙伴的鉴定

• 批准号: 6499604
• 负责人: GREGORY G. GERMINO
• 金额: $ 12.41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499604
• 关键词: antibody formation; binding sites; calcium channel; cell line; confocal scanning microscopy; embryo /fetus cell /tissue; gene interaction; genetic library; genetic models; glycoproteins; immunofluorescence technique; immunoprecipitation; laboratory mouse; mature animal; membrane proteins; molecular cloning; mutant; polycystic kidney; protein binding; protein localization; protein protein interaction; protein structure function; yeast two hybrid system

This is a competitive renewal of a project (DK51042) that was submitted in response to an RFA on PKD in 1995. We hypothesized that the three forms of ADPKD were likely to result from defects in interactive factors involved in a common pathway. We based this prediction on the observation that all forms of ADPKD has clinically indistinguishable presentations. Moreover, we predicted that the respective gene products were likely to be closely opposed on the pathway. The first submission proposed to use a number of complementary strategies to identify and characterize protein binding partners of PKD1, the protein most commonly mutated in ADPKD. We initiated our studies using the C-terminus of PKD1 to screen a yeast two- hybrid library. This work resulted in the discovery of an important, previously unrecognized structural feature of the PKD1 C-terminus. We have since shown that this coiled coil structure is capable of mediating direct interactions between PKD1 and the PKD2 gene product. Our library screening resulted in the isolation of 5 independent, overlapping clones of a Dbl like gene, P-CIP1, that interacted with high specificity to PKD1. Further study, suggested that a very similar gene, Trio, has an expression pattern that more closely overlaps that of PKD1. We found that Trio also interacts with PKD1 with great specificity. In vivo studies have shown that the relevant domains of the two proteins are capable of binding under physiologic conditions. In the present application, we seek to determine the biological relevance of the previously observed interactions. Specifically, we will use a novel cell line generated in the laboratory that has stable expression of full length PKD1 and a battery of well qualified antisera to demonstrate in vivo interactions of full length or native proteins. We also will test for functional consequences of the interactions. Finally, we propose to continue the search for PKD1 binding partners. We will screen a 14.5- 15.5 murine fetal cDNA library using the yeast two hybrid system. Positive clones will be thoroughly evaluated suing the strategies and reagents previously used the strategies and reagents previously used to characterize PKD1 and Trio. These studies will complement the efforts of other Center investigators and provide new insights into the pathways regulated by PKD1.

这是一个项目 (DK51042) 的竞争性更新，该项目是针对 1995 年关于 PKD 的 RFA 提交的。我们假设 ADPKD 的三种形式可能是由共同途径中涉及的相互作用因素的缺陷引起的。我们基于以下观察得出这一预测：所有形式的 ADPKD 都具有临床上无法区分的表现。此外，我们预测各自的基因产物可能在该途径上是紧密相反的。第一份提交的材料建议使用多种互补策略来识别和表征 PKD1 的蛋白质结合配偶体，PKD1 是 ADPKD 中最常见突变的蛋白质。我们开始使用 PKD1 的 C 末端来筛选酵母双杂交文库。这项工作发现了 PKD1 C 末端的一个重要的、以前未被识别的结构特征。我们已经证明这种卷曲螺旋结构能够介导 PKD1 和 PKD2 基因产物之间的直接相互作用。我们的文库筛选分离出 Dbl 样基因 P-CIP1 的 5 个独立、重叠的克隆，该基因与 PKD1 具有高度特异性相互作用。进一步的研究表明，一个非常相似的基因 Trio 的表达模式与 PKD1 的表达模式更接近。我们发现 Trio 还与 PKD1 相互作用，具有很强的特异性。体内研究表明，两种蛋白质的相关结构域能够在生理条件下结合。在本申请中，我们试图确定先前观察到的相互作用的生物学相关性。具体来说，我们将使用实验室生成的新型细胞系，该细胞系稳定表达全长 PKD1 和一系列合格的抗血清，以证明全长或天然蛋白质的体内相互作用。我们还将测试交互的功能后果。最后，我们建议继续寻找 PKD1 结合伙伴。我们将使用酵母二杂交系统筛选 14.5-15.5 鼠胎儿 cDNA 文库。将使用之前用于表征 PKD1 和 Trio 的策略和试剂对阳性克隆进行彻底评估。这些研究将补充其他中心研究人员的努力，并为 PKD1 调节的途径提供新的见解。