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J. Biol. Chem., Vol. 278, Issue 24, 22079-22089, June 13, 2003

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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^*

Mohamed Benharouga  , Manu Sharma  ¶, Jeffry So  ||, Martin Haardt , Luke Drzymala , Milka Popov  **, Blanche Schwapach , Sergio Grinstein , Kai Du  and Gergely L. Lukacs  

From the Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada, ZMBH, INF 282, D-69120 Heidelberg, Germany

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 26 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 26 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 26 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.

Received for publication, January 30, 2003

^* This work was supported by grants from the Canadian Cystic Fibrosis Foundation (CCFF), the Canadian Institute of Health Research (CIHR), and the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by a CCFF postdoctoral fellowship. Present address: Laboratoire de Biophysique Moléculaire et Cellulaire, Département de Biologie Moléculaire et Structurale, Commissariat à l'Energie Atomique-Grenoble, 17 Rue des Martyrs, F-38054, Grenoble cedex 09, France.

^¶ Supported by a CIHR doctoral studentship.

^|| Recipient of a CCFF summer studentship.

^** Supported in part by a CIHR postdoctoral fellowship.

To whom correspondence may be addressed: Hospital for Sick Children Research Institute, Program in Cell Biology, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5125; Fax: 416-813-5771; E-mail: glukacs{at}sickkids.ca.

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