Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal

doi:10.1074/jbc.274.6.3602

Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal*

From the Department of Cell Biology, the Gregory Fleming James Cystic Fibrosis Research Center, and the ^§Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

Abstract

We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192–5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2–78) and carboxyl-terminal tail regions (residues 1391–1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2–78) and CFTR-(1391–1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440–1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had ∼40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.

Footnotes

↵* This work was supported by NIDDK, National Institutes of Health, Grant R29-DK47339 Cystic Fibrosis Foundation Grant COLLAW96PO.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.
↵‡ Present address: 500 EMRB, University of Iowa, Iowa City, IA 52242.
↵¶ To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama at Birmingham, BHSB 392, UAB Station, Birmingham, AL 35294-0005. Tel.: 205-934-1002; Fax: 205-975-5648; E-mail: jcollawn{at}uab.edu.
Abbreviations:

CFTR

cystic fibrosis transmembrane conductance regulator

TR, transferrin receptor

SPQ

6-methoxy-N-(3-sulfopropyl)quinolinium

BSA

bovine serum albumin

PBS

phosphate-buffered saline.
- Received August 21, 1998.
- Revision received October 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.