The Down Regulated in Adenoma (dra) Gene Product Binds to the Second PDZ Domain of the NHE3 Kinase A Regulatory Protein (E3KARP), Potentially Linking Intestinal Cl-/HCO3- Exchange to Na+/H+ Exchange

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.