NDFIP1: the missing adaptor for aquaporin‐2 regulation by NEDD4 and NEDD4L (LB723)

Abstract

The aquaporin 2 (AQP2) water channel in collecting duct principal cells is essential for renal water reabsorption. Fine‐tuning of its expression in the apical membrane is critical for reabsorption of the proper amount. This fine‐tuning is the result of signals regulating AQP2 exocytosis and endocytosis. Whereas vasopressin‐induced cAMP signaling causes AQP2 translocation to the apical membrane, protein kinase C‐activating hormones stimulate ubiquitination‐dependent internalization of AQP2. The involved E3‐ubiquitin ligase has not been identified yet.HEK293 cells (from human embryonic kidney) and mpkCCD cells (from murine cortical collecting duct) were used to study ubiquitination and degradation of AQP2.SiRNA knockdown of NEDD4 and NEDD4L in mpkCCD cells increased AQP2 abundance, while both proteins did not interact with AQP2. Yeast Two‐Hybrid assay using full‐length AQP2 as bait identified NEDD4 family interacting protein 2 (NDFIP2). NDFIP2 and its homologue NDFIP1 are adaptor proteins, which bind NEDD4 ubiquitin ligases via their PY‐motifs and bring them close to target proteins. In HEK293 cells, NDFIP1/2 bound AQP2 and were essential for NEDD4/NEDD4L‐mediated ubiquitination and degradation of AQP2, an effect not observed with PY‐lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance, but did not affect the extent of AQP2 internalization. In mouse kidney, NDFIP1, but not NDFIP2, co‐localized and co‐immuno‐precipated with AQP2.Our results reveal NDFIP1 as a potent and rate‐limiting regulator of NEDD4/NEDD4L‐mediated ubiquitination and degradation of AQP2.Grant Funding Source: Supported by a Marie Curie Intra European Fellowship grant for CT and a VICI grant for PD.