IDENTIFICATION OF PKD1 PROTEIN BINDING PARTNERS

这是一个竞争性更新的项目（DK51042），是根据1995年关于PKD的RFA提交的。我们假设这三种形式的ADPKD可能是由于共同途径中涉及的相互作用因子的缺陷造成的。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可能在该途径上紧密相反。第一份报告提出使用一些互补策略来鉴定和表征PKD1的蛋白结合伴侣，PKD1是ADPKD中最常见的突变蛋白。我们首先利用PKD1的c端筛选酵母双杂交文库。这项工作的结果是发现了PKD1 c端一个重要的、以前未被认识到的结构特征。我们已经证明这种卷曲的线圈结构能够介导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分离出了5个独立的重叠克隆，它们是一个类似于Dbl的基因P-CIP1，对PKD1具有高特异性。进一步的研究表明，一个非常相似的基因Trio具有与PKD1更紧密重叠的表达模式。我们发现Trio也与PKD1有很强的特异性相互作用。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。在目前的应用中，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，我们将使用在实验室中生成的具有稳定表达全长PKD1和一系列合格的抗血清的新细胞系来证明全长或天然蛋白的体内相互作用。我们还将测试交互的功能结果。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小鼠胎儿cDNA文库。阳性克隆将使用先前用于表征PKD1和Trio的策略和试剂进行彻底评估。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.