Forward trafficking of NPT2a in the renal proximal tubule is inhibited by increased association of intracellular proteins identified by proteomic analysis

Abstract

NHERF1 (Na‐H Exchanger Regulator Factor 1) is a PDZ protein critical for apical membrane trafficking of NPT2a through interaction with the C terminal PDZ binding motif (TRL) of NPT2a. We previously demonstrated that NPT2a constructs lacking TRL traffic appropriately to the plasma membrane in HEK293 cells. This suggested that additional proximal tubule cell‐specific mechanisms regulate NPT2a membrane expression. Proteomic analysis of NPT2a immunoprecipitated proteins from wild type and NHERF1 KO kidneys identified several proteins with an increased NPT2a association in NHERF1 KO kidneys. We hypothesized that in NHERF1 KO proximal tubule cells, forward trafficking of NPT2a is inhibited by increased intracellular association with one or more of these proteins.To test our hypothesis, we generated cDNA constructs encoding: NPT2a full‐length (FL); NPT2a PDZ1‐deficient (TRL−); and a NPT2a/NHERF1 chimera wherein the EBD of NHERF1 replaces the TRL motif of NPT2a (CH). We transfected opossum kidney (OK) and NHERF1‐deficient opossum kidney (OKH) cells with these wild‐type and mutant NPT2a constructs and assessed their expression with confocal microscopy. We determined the OK/OKH cellular expression levels of the NPT2a immunoprecipitated proteins identified by proteomic analysis by western blot analysis. We also examined in vivo expression of NPT2a and 14‐3‐3 epsilon in wild type and NHERF1 KO kidney with immunofluorescence microscopy.Expression of NPT2a TRL− in OK cells or expression of NPT2a FL in OKH cells resulted in increased intracellular NPT2a expression. Transfection of the NPT2a CH in OK and OKH cells both showed a pattern of combined plasma membrane and intracellular expression that was similar to that seen in the OK cells expressing NPT2a TRL−. Comparison of NPT2a immunoprecipitated proteins in wild type and NHERF1 KO mice identified increased NPT2a association with calreticulin, 14‐3‐3 epsilon, beta catenin 1, and clathrin. Interestingly, western blot analysis demonstrated similar expression levels of calreticulin and 14‐3‐3 epsilon in OK and OKH cells, but markedly reduced expression of beta catenin 1 and clathrin in OKH cells. In silico analysis identified 5 putative 14‐3‐3 epsilon binding motifs within NPT2a, so we examined the localization of NPT2a and 14‐3‐3 epsilon in wild type and NHERF1 KO mouse kidney. Confocal microscopy demonstrated apical and sub‐apical expression of NPT2a in wild type and NHERF1 KO mice, respectively. However, no co‐localization of 14‐3‐3 epsilon with NPT2a was observed.Transfection of wild type and mutant NPT2a constructs in OK/OKH cells support more than a membrane anchoring role for NHERF1. 14‐3‐3 epsilon expression does not co‐localize with NPT2a in NHERF1 KO kidneys, suggesting an indirect role for 14‐3‐3 epsilon in the regulation of NPT2a trafficking.Support or Funding InformationSupport for this research provided by the University of Louisville (Department of Medicine) and the Robley Rex Veteran Affairs Medical Center Clinical Research Fund to EDL.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.