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.

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Johns Hopkins University
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