The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors.
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 vivo cross-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 Tyr(1424) is present in a beta-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.
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
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D014443:Tyrosine
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; C505032:CFTR protein, human; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014443:Tyrosine