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J. Biol. Chem., Vol. 279, Issue 21, 22578-22584, May 21, 2004

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Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines^*

Károly Varga, Asta Jurkuvenaite, John Wakefield¶, Jeong S. Hong, Jennifer S. Guimbellot||, Charles J. Venglarik, Ashutosh Niraj, Marina Mazur, Eric J. Sorscher**, James F. Collawn, and Zsuzsa Bebök

From the Departments of Cell Biology, ^**Medicine, ^||Genetics and Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the ^¶Tranzyme Corporation, Birmingham, Alabama 35209

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, 75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are 4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.

Received for publication, February 11, 2004 , and in revised form, March 22, 2004.

^* This work was supported in part by a fellowship from the Research Development Program of the Cystic Fibrosis Foundation (CFF) (to K. V.), an American Lung Association grant (to Z. B.), National Institutes of Health Grant DK60065 (to J. F. C.), and a grant from the Research Development Program of the CFF and the National Institutes of Health (to E. J. S.). 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.

This study represents a collaborative effort between the Bebõk and Collawn laboratories, and both investigators contributed equally to the supervision of the experiments.

To whom correspondence should be addressed: Dept. of Cell Biology and Gregory Fleming James CF Research Center, University of Alabama at Birmingham, MCLM 760, 1918 University Blvd., Birmingham, AL 35294-0005. Tel.: 205-975-5449; Fax: 205-934-7593; E-mail: bebok{at}uab.edu.

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