Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 pr otein complexes. Hybridomas were selected based on ability to immunoprecipi tate NHE3, One of the resulting mAbs (10A3) labeled a high molecular mass ( >200 kDa) protein and stained primarily the coated pit region of the proxim al tubule in a manner similar to that described for megalin (gp330). We the n confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipita ted and immunoblotted the same protein, namely megalin. mAb 10A3 specifical ly co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal mem branes, indicating specificity of the NHE3-megalin interaction. When immuno precipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centr ifugation, we found that NHE3 exists in two states with distinct sedimentat ion coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-ter minal 131 amino acids of NHE3 are blocked. Taken together, these findings i ndicate that a significant pool of NHES exists as a multimeric complex with megalin in the brush border of the proximal tubule.