Co-immunoprecipitation of a 24 kda protein with nhe-1, the
ubiquitous isoform of the na+/h+ exchanger.
Goss, Greg, John Orlowski, and Sergio Grinstein.
DIVISION OF CELL BIOLOGY, HOSPITAL FOR SICK CHILDREN, 555
UNIVERSITY AVE, TORONTO, M5G 1X8 CANADA AND, DEPARTMENT OF
PHYSIOLOGY, McGILL UNIVERSITY, MONTREAL, CANADA
APStracts 2:0393C, 1995.
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 NHE-1 with a protein of nearly equal to 24
kDa (p24). Immunoprecipitation of NHE-1 from lysates of
[35S]cysteine/methionine labeled cells using an anti-NHE-1 antibody
demonstrated specific co-immunoprecipitation of p24 with NHE-1. The
stoichiometry of p24 relative to NHE-1, assessed by their radiolabel
content, was consistent between experiments and among cell types.
Immunoblotting demonstrated that p24 is not a proteolytic product of
NHE-1. Internal deletion mutants and chimeras of NHE-1/NHE-3 suggest
that p24 binds to residues 515-566 or 695-815 of NHE-1, or to the
transmembrane region of both NHE-1 and NHE-3. p24 is not
constitutively phosphorylated nor could phosphorylation be induced by
serum/phorbol ester treatment. p24 binding to NHE-1 is Ca2+
-independent. p24 failed to bind [[gamma]-32P]GTP in a blot overlay
assay suggesting it is not a small molecular weight GTP-binding
protein. The identification of the p24:NHE-1 interaction may
contribute to our understanding of antiporter regulation.
Received 27 July 1995; accepted in final form 26 October 1995.
APS Manuscript Number C459-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 November 95