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Binding of Calcineurin Homologous Protein Isoforms 1 and 2 for the Na/H+ Exchanger Isoform 1 Using Recombinant Fluorescent Proteins
Author(s) -
Fray Emily,
Wallert Mark,
Provost Joseph
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.897.25
Subject(s) - gene isoform , biochemistry , intracellular ph , transfection , chemistry , recombinant dna , microbiology and biotechnology , biology , intracellular , gene
The sodium hydrogen exchanger isoform 1 (NHE1) is a transmembrane transport protein critical for cell motility and intracellular pH regulation. NHE1 is regulated by a variety of cofactors and binding partners, including calcineurin homologous protein (CHP). Isoform 1 of CHP (CHP1) is expressed ubiquitously and is required for basal NHE1 activity; however, isoform 2 (CHP2) expression is only detected in transformed cancer cells and has been implicated as a putative target for cancer therapies. We have expressed and purified recombinant‐affinity tagged c‐terminal tail of NHE, RFP‐CHP1 and GFP‐CHP2 and used these for in vitro capture assays. This work demonstrates the affinity of each CHP isoform with NHE1. In vivo , we have stably expressed full‐length GST‐tagged NHE1 in lung fibroblast cells (PS120). Using pull‐down assays in cells transiently transfected with CHP1 or CHP2 we further demonstrate the affinity of CHP1 and CHP2 with NHE1. Follow‐up experiments with altered NHE1 amino acids are used to define potential sites of interaction of CHP2 and NHE1. Together this data illustrates the different affinity of CHP2 for NHE1 binding and points to a potential regulatory site for NHE1 function.