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Papers In Press, published online ahead of print September 17, 2001
Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pitttsburgh, PA 15261
Corresponding Author: nabrad{at}pitt.edu
The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, Y1424DSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes has been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the m subunit of AP-2. Crosslinking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides crosslinked a 50 kDa subunit of purified AP-2 complexes, the apparent molecular weight of m2. Furthermore, isolated m2 bound to the sorting motif, YDSI, both in crosslinking experiments and GST-pulldown experiments, confirming that m2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative m2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared to wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the m2 subunit of AP-2 and mutations in m2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.
J. Biol. Chem, 10.1074/jbc.M104545200
Submitted on May 18, 2001
Revised on August 27, 2001
Accepted on September 14, 2001
mu2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin mediated endocytic pathway
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