The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane

Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithel ial cells is critical for vectorial chloride transport. Previously, we repo rted that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interac tion with the PDZ domain-containing protein EBP50 (NHERF). PDZ interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to Identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride se cretion. Point substitution of the C-terminal leucine (Leu at position 0) w ith alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chlori de secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but re duced interaction between CFTR and EBP50, efficient expression of CFTR in t he apical membrane as well as chloride secretion. By contrast, individual p oint substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on me asured parameters. We conclude that the PDZ-interacting domain, in particul ar the leucine (position 0) and threonine (position -2) residues, are requi red for the efficient, polarized expression of CFTR in the apical plasma me mbrane, interaction of CFTR with EBP50, and for the ability of CFTR to medi ate chloride secretion, Mutations that delete the C terminus of CFTR may ca use cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.