Mutational analysis of calcineurin B homologous protein isoform 2 binding to the Na⁺ ‐ H⁺ exchanger isoform 1 (NHE1): NHE1 mutations H523I and H523G (796.7)

Abstract

The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a spectrum of function in cells from solid tumors including cell proliferation, adhesion, migration and invasion. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors for NHE1 function. Both CHP1 and CHP 2 bind to the same domain on NHE1, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells activation of NHE1, leading to enhances tumorigenic behavior. We will investigate the ability of mutations to a key amino acid in the binding domain (H523) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, H523I and H523G, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct as well as how changes in CHP2 binding alters adhesion, proliferation, and migration in cells expressing the NHE1 mutations relative to cells expressing wild‐type NHE1.