COIMMUNOPRECIPITATION OF A 24-KDA PROTEIN WITH NHE1, THE UBIQUITOUS ISOFORM OF THE NA+ H+ EXCHANGER/

Ancillary proteins have been proposed to account for phosphorylation-i ndependent regulation of the Na+/H+ exchanger (NHE), but such putative proteins have not been identified. Here we describe the specific asso ciation of NHE1 with a protein of similar to 24 kDa (p24). Immunopreci pitation of NHE 1 from lysates of [S-35]cysteine- and/or methionine-la beled cells with the use of an anti-NHE1 antibody demonstrated specifi c coimmunoprecipitation of p24 with NHE1. The stoichiometry of p24 rel ative to NHE1, assessed by their radiolabel content, was consistent be tween experiments and among cell types. Immunoblotting demonstrated th at 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 NHE 3. Protein p24 is not constitutively phosphorylated nor could phosphor ylation be induced by serum or phorbol ester treatment. Binding of p24 to NHE1 is Ca2+ independent. Protein p24 failed to bind [gamma-P-32]G TP 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.