PROJECT ABSTRACT/SUMMARY Understanding how mutations in PKD1 and TRPP2 lead to Autosomal Dominant Polycystic Kidney Disease remains a major obstacle in the search for effective therapies to treat this prevalent human disease. The protein products of these genes (PKD1 and TRPP2) form a receptor-channel complex for which the activation mechanism remains unknown. For this reason, investigations targeting the downstream effects of dysfunctional complexes continue to pose a challenge. TRPP2 functions as a nonselective cation channel yet the functions ascribed to PKD1 and to PKD1/TRPP2 as a complex are incompletely understood in an integrated or disease- relevant perspective. PKD1 has been shown to form an ion channel subunit in the 1:3 PKD1/TRPP2 complex, and independently has been proposed to act as an atypical G protein-coupled receptor. WNT ligands have recently been shown to activate the PKD1/TRPP2 complex leading to TRPP2-mediated currents. This finding presents a novel opportunity to investigate the mechanism of PKD1/TRPP2 activation and the role of PKD1. The objective of the proposed research is to resolve the WNT-induced activation mechanism of the PKD1/TRPP2 complex. This objective will be attained by testing the hypothesis that WNT binding to PKD1 leads to the downstream activation of TRPP2 via intermediary G protein signaling. The rationale is that information from these studies will yield insight into PKD1/TRPP2 function and regulation. This knowledge will drive future investigations to determine how mutations that compromise function can lead to cystic kidney disease. To test the hypothesis, the following three specific aims will be pursued. Aim 1 will test if WNT binding to PKD1 is necessary and sufficient for PKD1/TRPP2 channel activation. Residues within the minimal WNT binding domain of PKD1 that are necessary for the interaction with WNT will be identified using pull-down and in situ binding assays. Patch clamp techniques will then be used to determine if mutations that disrupt WNT binding subsequently disrupt WNT-induced channel activation. Aim 2 will investigate the role of WNT in the function of PKD1 as an atypical G protein-coupled receptor. This will be accomplished using NanoBRET assays to determine whether WNT ligands induce PKD1-mediated G protein signaling and if WNT binding to PKD1 induces PKD1 receptor desensitization. Aim 3 will determine the role of PKD1-mediated G protein signaling in TRPP2 activation. This will be achieved using NanoBRET and patch clamp to test if loss of PKD1-mediated G protein signaling disrupts channel activation, investigate the association of G proteins with TRPP2, and determine if inhibition of G protein signaling prevents WNT-induced TRPP2 activity. Proposed studies will provide a unique training opportunity in biochemistry, molecular biology, GPCR and ion channel function, in the context of one of the most common genetic diseases. The successful completion of this proposal will yield a defined ...