Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.

Prince LS, Peter K, Hatton SR, Zaliauskiene L, Cotlin LF, Clancy JP, Marchase RB, Collawn JF
J Biol Chem. 1999 274 (6): 3602-9

PMID: 9920908 · DOI:10.1074/jbc.274.6.3602

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 approximately 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.

MeSH Terms (13)

Amino Acid Sequence Animals Base Sequence COS Cells Cystic Fibrosis Transmembrane Conductance Regulator DNA Primers Endocytosis Molecular Sequence Data Protein Sorting Signals Receptors, Transferrin Recombinant Fusion Proteins Sequence Homology, Amino Acid Tyrosine

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