IDENTIFICATION OF PKD1 PROTEIN BINDING PARTNERS

PKD1 蛋白结合伙伴的鉴定

• 批准号: 6349101
• 负责人: GREGORY G. GERMINO
• 金额: $ 28.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349101
• 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（DK 51042）的竞争性延期，该项目是1995年根据PKD RFA提交的。我们假设这三种形式的ADPKD可能是由共同途径中涉及的相互作用因子的缺陷引起的。我们的预测是基于观察到所有形式的ADPKD在临床上都有难以区分的表现。此外，我们预测，各自的基因产物很可能是密切反对的途径。第一份提交材料建议使用一些互补策略来鉴定和表征PKD 1的蛋白结合伴侣，PKD 1是ADPKD中最常见的突变蛋白。我们利用PKD 1的C-末端来筛选酵母双杂交文库。这项工作发现了PKD 1 C-末端的一个重要的、以前未被认识的结构特征。我们已经证明，这种卷曲螺旋结构能够介导PKD 1和PKD 2基因产物之间的直接相互作用。我们的文库筛选导致分离出5个独立的、重叠的Dbl样基因P-CIP 1克隆，其与PKD 1具有高特异性相互作用。进一步的研究表明，一个非常相似的基因Trio的表达模式与PKD 1的表达模式更紧密地重叠。我们发现Trio也与PKD 1具有很强的特异性相互作用。体内研究表明，这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相关性。具体而言，我们将使用一种新的细胞系在实验室中产生的全长PKD 1和一组合格的抗血清，以证明在体内的全长或天然蛋白质的相互作用的稳定表达。我们还将测试相互作用的功能后果。最后，我们建议继续寻找PKD 1结合伙伴。我们将利用酵母双杂交系统筛选14.5- 15.5的小鼠胎儿cDNA文库。将使用先前用于表征PKD 1和Trio的策略和试剂对阳性克隆进行全面评价。这些研究将补充其他中心研究人员的努力，并为PKD 1调控的途径提供新的见解。