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J. Biol. Chem., Vol. 269, Issue 4, 2589-2596, Jan, 1994
P Fafournoux, J Noel and J Pouyssegur
In this study, we have investigated whether members of the Na+/H+ exchanger
(NHE) family are oligomers and whether such oligomeric structure is
required for function. Fibroblasts overexpressing NHE1 were treated briefly
at 0 degrees C with the cross-linker disuccinimidyl suberate, then
membranes were prepared and proteins analyzed by SDS-polyacrylamide gel
electrophoresis. Disuccinimidyl suberate treatment converted quantitatively
the immunoreactive monomeric form of NHE1 (110 kDa) to a putative dimeric
form (210 kDa). Utilization of NHE1 mutant deleted of the cytoplasmic
domain (delta 515TH) demonstrates that the transmembrane domain of the
antiporter is sufficient for dimerization. Moreover, coimmunoprecipitation
of NHE1 and delta 515TH, coexpressed in the same cell, formally proved the
existence of dimers. This dimerization was also shown to take place with
the epithelial and apically expressed NHE3 isoform, suggesting that
oligomerization is a common feature of these transporters. However,
coexpression of NHE1 and NHE3 in the same cells did not lead to the
formation of heterodimers demonstrating an isoform specificity for the
subunit interaction. The domain(s) involved in the isoform- specific
dimerization is (are) likely to be confined within the transmembrane
segments, as deletion of the 300 amino acids of the cytoplasmic domain did
not disrupt dimerization. Exploiting the dimeric properties of the receptor
tyrosine kinases and the fact that dimerization triggers kinase activity,
we constructed a NHE1/insulin receptor chimera to probe NHE1 dimerization
in vivo. When transfected into hamster fibroblasts, this chimera containing
the N-terminal transmembrane domain of NHE1 and the cytoplasmic
beta-subunit domain of the insulin receptor generates a functional
transporter that is autophosphorylated on tyrosine and that presents
properties of a constitutively active insulin receptor. These findings
support the notion that NHE1 exists in an oligomeric state in intact cells.
Finally, to test whether individual subunits of NHE1 are the minimum
functional unit for Na+/H+ exchange, we coexpressed a truncated form of
NHE1 (delta 515) together with an inactive mutant of NHE1 (E262I). In spite
of good expression of the inactive transporter and its capacity to dimerize
with active NHE1, no dominant negative effect was observed on
amiloride-sensitive 22Na+ flux. This observation would suggest that
individual subunits of NHE1 function independently within the oligomeric
state.
Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers
Centre de Biochimie, Centre National de la Recherche Scientifique, Universite de Nice, Parc Valrose, France.
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