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

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic genetic disorders, caused by mutations in receptor PKD1 or ion channel PKD2, and is characterized by progressive renal cyst development with additional hepatic and extrarenal manifestations. As effective treatments for ADPKD remain limited, further investigation into the function and regulation of PKD proteins is needed. Using biotin-based proximity labeling combined with mass spectrometry in human embryonic kidney (HEK) cells, here we identify Ras association domain family member 4 (RASSF4) as a potential PKD2-interacting protein. The association between PKD2 and RASSF4 is validated by co-immunoprecipitation, bimolecular fluorescence complementation, and in vitro binding assays in HEK cells and mouse kidneys. Functional analyses using two-electrode voltage clamp electrophysiology in Xenopus oocytes demonstrate that RASSF4 enhances PKD2 channel activity without affecting its membrane expression. In vivo studies in larval zebrafish show that RASSF4 over-expression alleviates, whereas Rassf4 knockdown exacerbates, Pkd2 knockdown-associated phenotypes, including tail curling, pronephric cyst formation, renal filtration defects, and motor dysfunction. Disruption of the RASSF4/PKD2 interaction using a blocking peptide (amino acid P134-S168) abolishes RASSF4-mediated stimulation of PKD2 channel function and phenotypic rescue, while worsening disease severity, likely by interfering with endogenous complex formation. Mechanistically, RASSF4 enhances the functionally critical intramolecular interaction between the PKD2 N- and C-termini and suppresses RAS/MAPK signaling in HEK cells. Together, these findings identify RASSF4 as a PKD2 regulator and suggest that the RASSF4/PKD2 complex represents a potential therapeutic target for ADPKD.