We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na ؉ /H ؉ exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene but the association between mutation (genotype) and disease presentation (phenotype) is not straightforward. We have been investigating whether variants in the CFTR gene that alter splicing efficiency of exon 9 can affect the phenotype produced by a mutation. A missense mutation, R117H, which has been observed in three phenotypes, was found to occur on two chromosome backgrounds with intron 8 variants that have profoundly different effects upon splicing efficiency. A close association is shown between chromosome background of the R117H mutation and phenotype. These findings demonstrate that the genetic context in which a mutation occurs can play a significant role in determining the type of illness produced.

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

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