Mice homozygous for the Oak Ridge Polycystic Kidney (orpk) mutation develop renal lesions resembling that seen in human patients with polycystic kidney disease (PKD). The gene (Tg737) responsible for the phenotype has been identified and characterized. The protein encoded by Tg737 contains ten copies of the tetratricopeptide repeat (TPR), a motif that is involved in protein-protein interactions. Using the yeast two-hybrid assay, we identified several proteins that bind to Tg737. Consistent with the orpk pathology, these proteins play important roles in cellular differentiation, epithelial polarity, and epidermal growth factor receptor (EGFR) signaling and stability. Most important, one of these cDNAs encodes a portion of polycystin-1. This interaction is noteworthy since mutations in polycystin-1 and Tg737 are directly associated with PKD in humans and mice, respectively. These data suggest that polycystin-1 and Tg737 are part of a common signaling pathway and that analysis of Tg737 will help elucidate the molecular basis of this human disease. In this regard, this proposal has three aims directed at characterizing the Tg737 protein, analyzing Tg737 protein interactions, and determining how disruption of the Tg737 pathway results in renal cyst formation. Specifically, in the first aim we will analyze Tg737 protein expression and localization in wild type kidneys. In the second aim, we will confirm the interaction of Tg737 with two of the proteins isolated in the two-hybrid screen, and identify the domains that mediate their interaction. This analysis will give insight into whether these proteins function as a complex or whether their interactions occur through a common Tg737 domain, and are thus mutually exclusive. The purpose of the third aim is to determine whether the loss of Tg737 in orpk mutant alters the expression or localization of these proteins impeding their function. As part of this aim, we will analyze aspects of the Tg737 signaling pathway in cell culture by disrupting the interaction between Tg737 and these two proteins, independently. To accomplish this, we will generate dominant negative mutations by expressing the interacting domains at high levels in IMCD cells (mouse collecting duct). Because IMCD cells express endogenous Tg737 and the two interacting proteins, we predict that this will """"""""titer-out"""""""" the protein that associates with that domain, preventing their normal localization and function. Due to orpk pathology and the known functions of the two interacting proteins, we will analyze the affect of these mutations on EGFr and polycystin-1 localization and stability. Overall, results generated from this proposal will give important insight into the biological function of Tg737 and how disruption of the Tg737/polycystin-1 signaling pathway results in the formation of renal cyst.
