Premium
Calcineurin Homologous Protein Isoforms 1 and 2 Binding to the Sodium Hydrogen Exchanger 1
Author(s) -
Marshall Chelsea,
Wallert Mark,
Provost Joseph John
Publication year - 2016
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.30.1_supplement.600.11
Subject(s) - chemistry , biochemistry , gene isoform , intracellular ph , sodium–hydrogen antiporter , microbiology and biotechnology , intracellular , sodium , biology , organic chemistry , gene
The Sodium Hydrogen Exchanger (NHE1) is a ubiquitously expressed protein, which regulates cell volume, intracellular pH, and motility. This antiporter exchanges an intracellular proton for an extracellular sodium and is regulated by a range of protein or lipid interactions and phosphorylations. Calcineurin Homologous Protein isoform 1 (CHP1) and isoform 2 (CHP2) both bind to essentially the same site on the carboxyl terminus of NHE1. However, while CHP1 and CHP2 share a 61% amino residue homology, they have different binding affinities for NHE1. Understanding how one isoform can bind NHE with greater affinity than another is of interest because CHP1 is required for basic NHE1 function and a physiological role of CHP2 has not been identified. To study the interaction of each CHP isoform with NHE1, we have generated a GST‐NHE1 fusion protein (aa 803–846) and His tagged CHP1 and 2. After expression and reconstitution of a thrombin cleaved NHE1 peptide with CHP we determined the interaction using pull‐down and thermal melt. To identify possible binding determinates on NHE1 for the each_CHP i soforms we mutated_eight residues of NHE1 thought to play a role binding CHP. These key residues, include: Asn519Ala, Asn519Asp, Ile518Gln/Ile522Gln, Il e 534Lys, Il e 537Lys, His523Gly, His523Ile, and Asp536Gly. Protein interactions with recombinant CHP1 or CHP2 with each mutant NHE1 peptide was determined using thermal denaturation, circular dichroism and other biophysical methods. This work will illuminate potential interaction sites on NHE1 for CHP, and demonstrate sites common and unique for both CHP isoforms.