C-terminal Truncations Destabilize the Cystic Fibrosis Transmembrane Conductance Regulator without Impairing Its Biogenesis

doi:10.1074/jbc.274.31.21873

C-terminal Truncations Destabilize the Cystic Fibrosis Transmembrane Conductance Regulator without Impairing Its Biogenesis

A NOVEL CLASS OF MUTATION*

From the Program in Cell Biology and Lung Gene Therapy, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario M5G 1X8 and the Department of Laboratory Medicine and Pathobiology and the ^‖Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Abstract

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.

Footnotes

↵* This work was supported by the Canadian Cystic Fibrosis Foundation, the Medical Research Council (MRC) of Canada, and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Instrumentation was covered in part by an Ontario Thoracic Society block term grant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to this study.
↵§ Supported in part by a Hospital for Sick Children Restracom fellowship.
↵¶ Postdoctoral fellow of the Canadian Cystic Fibrosis Foundation.
↵** A Scholar of MRC Canada. To whom correspondence should be addressed: Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Canada. Tel:. 416-813-5125; Fax: 416-813-5771; E-mail: glukacs@sickkids.on.ca.
2 C. Taylor (University of Sheffield, UK), personal communication.
↵3 N. Kartner, Z. Grzelczak, and J. R. Riordan, unpublished observations.
↵4 Lowering the copy number and the expression of WT CFTR was achieved using 10 μmmethotrexate during the clonal selection of CHO-BQ1 cells. To assure a high copy number of WT and mutant CFTR, BHK-21 cells were selected in medium supplemented with 500 μm methotrexate.
Abbreviations:

CF

cystic fibrosis

ER

endoplasmic reticulum

CFTR

cystic fibrosis transmembrane conductance regulator

NBD

nucleotide binding domain

PKA

protein kinase A

WT

wild type

mAb

monoclonal antibody

DMEM

Dulbecco’s modified Eagle’s medium

IBMX

3-isobutylmethyl-1-xanthine

CTP

8-(4-chlorophenylthiol)

HA

hemagglutinin

NHERF

Na⁺/H⁺ exchanger regulatory factor
- Received March 16, 1999.
- Revision received May 24, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.