Regulation of the PKD2 channel function and associated disease phenotypes by RASSF4

ADPKD is caused by mutations in receptor PKD1 and ion channel PKD2. As no effective treatment is available for ADPKD, further studies on the function and regulation of PKD1 and PKD2 are required. Using biotin-neighborhood labeling in HEK293T cells and mass spectrometry analysis, we identified Ras association domain family member-4 (RASSF4) as a potential PKD2-binding protein. Our co-immunoprecipitation and in vitro binding assays confirmed the RASSF4/PKD2 association. Using the two-electrode voltage clamp electrophysiology in Xenopus oocytes, we showed that RASSF4 increases the PKD2 channel function without affecting its membrane expression. Consistently, in larval zebrafish, RASSF4 alleviated the severity of tail curling, pronephric cyst occurrence and swimming defects associated with PKD2 knockdown. We found that the RASSF4/PKD2 binding, which can be disrupted by RASSF4 blocking peptide P134-S168, is required for RASSF4 to stimulate the PKD2 channel function in oocytes and alleviate the disease phenotypes in larval zebrafish due to PKD2 knockdown. Further, we found that RASSF4 enhances the functionally critical intramolecular interaction between the PKD2 N- and C-termini and down-regulates the RAS/MAPK signaling. Thus, this study revealed RASSF4 as a novel PKD2 regulator that enhances its channel function and alleviates PKD2-associated disease phenotypes in zebrafish, presumably through suppressing the RAS/MAPK pathway.