The Carboxyl Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Binds to AP-2 Clathrin Adaptors*
- From the Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid and efficient endocytosis. Since functionally active CFTR is found in purified clathrin-coated vesicles isolated from both cultured epithelial cells and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivocross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carboxyl terminus of CFTR (amino acids 1404–1480) were also able to bind AP-2 but did not bind the Golgi-specific adaptor complex AP-1. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Secondary structure predictions suggest that Tyr1424 is present in a β-turn conformation, a conformation disrupted by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate efficient entry of CFTR into clathrin-coated vesicles.
Footnotes
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↵* This work was funded by grants from the Cystic Fibrosis Foundation and the National Institutes of Health (DK47850).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.
The amino acid sequence of CFTR can be accessed through NCBI Protein Database under NCBI accession numbers (human), AAC14012 (monkey), Q0055 (sheep), AAC48608 (rabbit), AAC60023 (frog), (mouse), (killifish), (cow), and (dogfish).
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↵‡ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-2845; Fax: 412-648-2844; E-mail: nabrad@pitt.edu.
- Abbreviations:
- CFTR
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cystic fibrosis transmembrane conductance regulator(s)
- GST
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glutathioneS-transferase
- PAGE
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polyacrylamide gel electrophoresis
- DTSSP
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3,3′-dithiobis[sulfosuccinimidyl]propionate
- Mes
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4-morpholineethanesulfonic acid
- GST-CT
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GST-carboxyl terminus
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- Received June 9, 1999.
- Revision received November 4, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
