Association of Na+-H+ exchanger isoform NHE3 and dipeptidyl peptidase TV in the renal proximal tubule.

In an attempt to identify proteins that assemble with the apical membrane N a+-H+ exchanger isoform NHE3, we generated monoclonal antibodies (in-Abs) a gainst affinity-purified NHE3 protein complexes isolated from solubilized r enal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the m-Abs (1D11) that specifically co-precipitated NHE3 but not villin or N aPi-2. Western blot analyses of microvillus membranes and immunoelectron mi croscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa prote in highly expressed on the apical membrane of proximal tubule cells. Immuno affinity chromatography was used to isolate the antigen against which mAb 1 D11 is directed. N-terminal sequencing of the purified protein identified i t as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.15), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contra st to the previously described NHE3-megalin complex, which principally resi des in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in wh ich NHE3 is active. Serial precipitation experiments confirmed that anti-me galin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Tak en together, these studies revealed an unexpected association of the brush border Na+-H+ exchanger NHE3 with dipeptidyl peptidase IV in the proximal t ubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.