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DRA (down regulated in adenoma) is an intestinal anion exchanger, acting in parallel with NHE3 to facilitate ileal and colonic NaCl absorption. Furthermore it is involved in small intestinal bicarbonate secretion. Because DRA has a PDZ interaction motif, which may influence its properties, we searched for DRA-interacting PDZ adapter proteins in the small intestine. Using an overlay assay with the recombinant DRA C-terminus as a ligand, a 70 kDa protein was labeled, which was restricted to the brush border membrane in rabbit duodenal and ileal mucosa and was not detected in the colon. Destruction of the C-terminal PDZ interaction motif abolished this band, suggesting a specific protein-protein interaction. The 70 kDa protein was identified as CAP70 (CFTR associated protein of 70 kDa) by an anti-CAP70 antibody and by two in vitro binding assays after cloning CAP70 from rabbit duodenum and ileum. The interaction was recapitulated in HEK cells transfected with DRA and PDZK1, the human orthologue of CAP70. Corresponding to the overlay assay, no CAP70 mRNA or protein was detected in the colon. In vitro protein-protein interaction studies revealed specific binding of DRA to the 2nd and 3rd PDZ domain, while CFTR is known to interact with PDZ1, PDZ3, and PDZ4. The composition of macromolecular complexes assembled by CAP70 in the distal small bowel is unknown. Its restricted expression shows that it cannot be involved in NaCl absorption in the proximal colon. We suggest that CAP70 mediates regulatory functions specific to the small intestine.

Intestinal electroneutral NaCl absorption is mediated by parallel operation of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange in the enterocyte apical membrane. The ion transporters involved are Na(+)/H(+) exchanger 3 (NHE3) and the down regulated in adenoma (dra) gene product. cAMP-mediated inhibition of NHE3 requires the transporter to bind to the second PDZ (PSD95, disk large, ZO1) domain of the adapter protein NHE3 kinase A regulatory protein (E3KARP). Because the C-terminal four amino acids of dra are ETKF (glutamate-threonine-lysine-phenylalanine), resembling a PDZ interaction motif, we hypothesized that dra may also bind to one of the PDZ domains of E3KARP. In vitro the ETKF motif of dra binds to the second PDZ domain of E3KARP, the affinity being comparable to that of the known ligand CFTR. The C-terminal phenylalanine, which is an unconventional residue in PDZ interaction motifs, can only be substituted by the classical residue leucine, but not by other hydrophobic residues (valine, isoleucine). Immunofluorescence colocalizes dra, NHE3, and E3KARP in the apical compartment of human proximal colon. We suggest a model in which both NHE3 and dra bind to the second PDZ domain of E3KARP and that linking of the transporters occurs through dimerization of E3KARP. In such a model, the first PDZ domain would remain available for instance for signal transduction proteins.