IDENTIFICATION OF PKD1 PROTEIN BINDING PARTNERS

这是一个项目(DK51042)的竞争性续展，该项目是对1995年关于PKD的RFA的回应而提交的。我们推测，这三种形式的ADPKD很可能是共同通路中涉及的交互因素的缺陷所致。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，在该途径上，各自的基因产物可能会紧密对立。第一份意见书建议使用一些补充策略来确定和表征PKD1的蛋白结合伙伴，PKD1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。这项工作的结果是发现了PKD1C-末端的一个以前未被发现的重要结构特征。自那以后，我们已经证明这种盘绕结构能够介导PKD1和PKD2基因产物之间的直接相互作用。我们的文库筛选结果是分离到5个相互独立的、重叠的DBL样基因P-CIP1，它们相互作用于PKD1。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的表达模式更紧密地重叠。我们发现Trio与PKD1的相互作用也具有很强的特异性。体内研究表明，这两种蛋白的相关结构域在生理条件下能够结合。在本申请中，我们寻求确定先前观察到的相互作用的生物学相关性。具体地说，我们将使用在实验室中产生的具有稳定表达全长PKD1的新型细胞系和一组质量良好的抗血清来演示全长或天然蛋白在体内的相互作用。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。我们将使用酵母双杂交系统筛选14.5-15.5个小鼠胎儿cDNA库。阳性克隆将使用以前使用的策略和试剂进行彻底评估，这些策略和试剂以前用于鉴定PKD1和Trio。这些研究将补充其他中心研究人员的努力，并为PKD1调控的途径提供新的见解。

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.