MOLECULAR MECHANISMS OF POLYCYSTIN FUNCTION

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Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease, with a gene frequency of 1 in 200-1,000 individuals, giving rise to approximately 6-9% of all end-stage renal disease. ADPKD is characterized by the growth of large epithelial-lined cysts from the nephrons and collecting ducts of affected kidneys. Approximately 85% of the cases of ADPKD are due to mutations in the PKD1 gene. The product of the PKD1 gene, polycystin-1, is thought to be a large, membrane-associated protein that may function as a receptor of adhesion molecule, possibly mediating cell-cell or cell-matrix interactions. The product of the PKD2 gene, polycystin-2 shows similarity to a family of calcium channel proteins and thus may be involved in calcium-mediated signal transduction. Polycystin-1 and -2 are also thought to interact with each other via the C-terminal cytosolic domains, raising the possibility that they function in a coordinated fashion to receive extracellular signals and transmit them to the cytoplasm. The functions of these proteins, however, are not known. Sequence analysis has revealed that the C-terminal cytosolic domains of polycystin-1 and -2 have a number of conserved motifs that suggest that these proteins function by mediating signal transduction. Thus, the central hypothesis of this proposal is that the functions of polycystin-2 and -2 are mediated by signal transduction and, as such, that these proteins are substrates for phosphorylation, that they interact with heterotrimeric G-proteins, and that polycystin phosphorylation regulates the interactions between polycystin-2 and -2 and between polycystin-2 and -2 and heterotrimeric G-proteins. This hypothesis will be addressed by the following specific aims: 1) The C-terminal cytosolic domains of polycystin-1 and polycystin-2 will be tested to determine if they are substrates for phosphorylation in vitro and in vivo, 2) The effects of phosphorylation of polycystin-1 and polycystin-2 on their interactions with each other will be determined in vitro and in vivo, and 3) Interactions between polycystin-1 and polycystin-2 and heterotrimeric G- proteins and the effects of polycystin phosphorylation on these interactions, will be tested in vitro and in vivo.