Autosomal Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 and PKD2 gene that encode polycystin 1 (PC1) and polycystin2 (PC2). PC2 is a non-selective Ca2+permeable channel, which may function in more than one subcellular location (plasma membrane, cilia, ER and Golgi), but the factors that regulate the dynamic translocation of PC2 between different cellular compartments are not well understood. Given that it is difficult to study PC2 function and signaling in humans or mice, alternative animal models are necessary to validate data derived from in vitro studies. The goal of this grant is to establish mechanisms for the dynamic translocation of human PC2 in zebrafish and medaka, to identify physiologically important protein partners of PC2 in the native cellular environment and to investigate possible signal transduction pathways regulating PC2 function. We have shown that PC2 Ser76 is phosphorylated by glycogen synthase kinase 3 (GSK3). In the presence of GSK3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment of MDCK cells but remains associated with the primary cilium. Co-injection of wild-type, but not a S76A/S80A mutant human PKD2 mRNA, rescued defects induced by disrupting the endogenous zebrafish pkd2 gene. We conclude that localization of PC2 is regulated by a unique GSK3 phosphorylation site both in vivo and in vitro. Based on this data, we hypothesize that PC2 function requires targeting to specific subcellular domains in addition to the cilium.
Our specific aims are: 1. To use zebrafish and medaka to identify human PC2 functional motifs required for correct subcellular targeting and normal PC2 function;2. to define function of two candidate PC2-interacting proteins PIGEA14 and PACS and 3. to establish that WNT, BMP dependent pathways are required for subcellular targeting of PC2. PC2 is functionally conserved among human, zebrafish and medaka. Therefore these studies will help elucidate PC2 function in normal human kidney and in pathological disease stages. These data will define pathways that regulate PC2 subcellular localization and function and identify novel therapeutic targets.
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