The B1 subunit of the H(+)ATPase is a PDZ domain-binding protein - Colocalization with NHE-RF in renal B-intercalated cells

The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amin o acid motif typical of PDZ-binding proteins that associate with the PDZ pr otein, NHE-RF (Na+/H+ exchanger regulatory factor). This B1 isoform is ampl ified in renal intercalated cells, which play a role in distal urinary acid -base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif, Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-trans ferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in viv o as part of the cytoplasmic VI portion of the H(+)ATPase, and the E subuni t was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a pept ide derived from the C terminus of the El but not the B2 isoform, NHE-RF co localized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-i ntercalated cells. In proximal tubules, NHE-RF was located in the apical br ush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, cons istent with the expression of the truncated B2 subunit isoform in this tubu le segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-int ercalated cells suggests a role in generating, maintaining, or modulating t he variable H(+)ATPase polarity that characterizes the B-cell phenotype.