Identification of proteins differentially associated with Npt2a in the presence and absence of NHERF1

Abstract

Background: NHERF1 deficient mice are hypophosphatemic and phosphaturic due to failure of functional apical membrane localization of Npt2a. Wild type (WT) and NHERF1 knock out (KO) mice show similar proximal tubule protein expression of Npt2a. Previous studies have shown that dissociation of Npt2a and NHERF1 stimulated by PTH and FGF23 results in lysosomal degradation of Npt2a. The persistence of Npt2a expression in NHERF1 deficiency suggests a defect in Npt2a forward traffcking. Hypothesis: We hypothesized that comparison of Npt2a-associated proteins from WT and KO mouse kidney cortex would identify novel proteins critical for forward traffcking of Npt2a. Methods: We immunoprecipitated homogenized kidney cortex from WT and NHERF1 KO C57Bl6 mice using a polyclonal Npt2a antibody followed by proteomic analysis using liquid chromatography-mass spectrometry and the STRING database of known and predicted protein-protein interactions. Results: We identified 31 proteins that showed statistically significantly greater association with Npt2a in WT kidney cortex and 50 proteins that showed statistically significantly greater association with Npt2a in KO kidney cortex. We did not identify greater association of Npt2a from KO kidney cortex with any of the other known PDZ proteins known to associate with Npt2a including PDZK1 and PIST. Among the proteins more highly associated with Npt2a in KO were 14-3-3 and valacyclovir hydrolase. STRING analysis of the WT-associated proteins identified membrane transport, phosphate ion homeostasis, and oxidative phosphorylation as important biological processes. Analysis of the KO-associated proteins identified DNA unwinding, regulation of cytoplasmic translation, and clathrin-dependent endocytosis were identified as important biological processes in the absence of NHERF1. Conclusions: We conclude that the aberrantly-traffcked Npt2a in KO has successfully traversed the Golgi and is correctly folded but may interact with 14-3-3 through its PDZ binding motif, preventing forward traffcking. We also conclude that the function of Npt2a may significantly change when the cotransporter does not properly forward traffc in the absence of NHERF1 expression. Support for EDL from Dept Veterans Affairs CX0001614 and the Pak Mineral Metabolism Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.