Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway.
The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, (1424)YDSI, 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 have 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 mu subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of mu 2. Furthermore, isolated mu 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that mu 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative mu 2 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 with 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 mu 2 subunit of AP-2 and mutations in mu 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.
Weixel
K M
KM
Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Bradbury
N A
NA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002966:Clathrin / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
C505032:CFTR protein, human; D002966:Clathrin; D004268:DNA-Binding Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014443:Tyrosine