The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia.

The conserved C-terminal peptide motif (1476DTRL) of the cystic fibrosis transmembrane conductance regulator (CFTR) ensures high affinity binding to different PSD-95/Disc-large/zonula occludens-1 (PDZ) domain-containing molecules, including the Na+/H+ exchanger regulatory factor (NHERF)/ezrin-radixin-moesin-binding phosphoprotein of 50 kDa. The physiological relevance of NHERF binding to CFTR is not fully understood. Individuals with mutations resulting in premature termination of CFTR (S1455X or Delta26 CFTR) have moderately elevated sweat Cl- concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Surprisingly, when expressed heterologously, the Delta26 mutation was reported to abrogate channel activity by destabilizing the protein at the apical domain and inducing its accumulation at the basolateral membrane (Moyer, B., Denton, J., Karlson, K., Reynolds, D., Wang, S., Mickle, J., Milewski, M., Cutting, G., Guggino, W., Li, M., and Stanton, B. (1999) J. Clin. Invest. 104, 1353-1361). The goals of this study were to resolve the contrasting clinical and cellular phenotype of the Delta26 CFTR mutation and evaluate the role of NHERF in the functional expression of CFTR at the plasma membrane. Complex formation between CFTR and NHERF was disrupted by C-terminal deletions, C-terminal epitope tag attachments, or overexpression of a dominant negative NHERF mutant. These perturbations did not alter CFTR expression, metabolic stability, or function in nonpolarized cells. Likewise, inhibition of NHERF binding had no discernible effect on the apical localization of CFTR in polarized tracheal, pancreatic, intestinal, and kidney epithelia and did not influence the metabolic stability or the cAMP-dependent protein kinase-activated chloride channel conductance in polarized pancreatic epithelia. On the other hand, electrophysiological studies demonstrated that NHERF is able to stimulate the cAMP-dependent protein kinase-phosphorylated CFTR channel activity in intact cells. These results help to reconcile the discordant genotype-phenotype relationship in individuals with C-terminal truncations and indicate that apical localization of CFTR involves sorting signals other than the C-terminal 26 amino acid residues and the PDZ-binding motif in differentiated epithelia.