AJP - Cell Physiology, Vol 270, Issue 5 C1493-C1502, Copyright © 1996 by American Physiological Society

ARTICLES

Coimmunoprecipitation of a 24-kDa protein with NHE1, the ubiquitous isoform of the Na+/H+ exchanger

G. Goss, J. Orlowski and S. Grinstein
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.

Ancillary proteins have been proposed to account for phosphorylation-independent regulation of the Na+/H+ exchanger (NHE), but such putative proteins have not been identified. Here we describe the specific association of NHE1 with a protein of approximately 24 kDa (p24). Immunoprecipitation of NHE1 from lysates of [35S] cysteine- and/or methionine-labeled cells with the use of an anti-NHE1 antibody demonstrated specific coimmunoprecipitation of p24 with NHE1. The stoichiometry of p24 relative to NHE1, assessed by their radiolabel content, was consistent between experiments and among cell types. Immunoblotting demonstrated that p24 is not a proteolytic product of NHE1. Internal deletion mutants and chimeras of NHE1/NHE3 suggest that p24 binds to residues 515-566 or 695-815 of NHE1 or to the transmembrane region of both NHE1 and NHE3. Protein p24 is not constitutively phosphorylated nor could phosphorylation be induced by serum or phorbol ester treatment. Binding of p24 to NHE1 is Ca2+ independent. Protein p24 failed to bind [gamma-32P]GTP in a blot-overlay assay, suggesting that it is not a low-molecular-weight GTP-binding protein. Identification of the p24:NHE1 interaction may contribute to our understanding of antiporter regulation.

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