Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-activated chloride channel that is localized to the plasma membrane and endosomal compartment. Endosomal targeting of CFTR is attributed to the Tyr(1424)-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr(1424) residue could partly inhibit the endocytosis of CFTR and its association with the adapter protein AP-2. To reveal additional endosomal targeting signals, site-directed mutagenesis of both a chimaera, composed of a truncated form of interleukin 2 receptor alpha chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assays revealed the presence of multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite endocytic signal, comprising a tyrosine (Tyr(1424)) and a di-Leu-based (Leu(1430)-Leu) motif. Whereas the replacement of any one of the three internalization motifs with alanine prevented the endocytosis of the TacT-Ct chimaera, mutagenesis of Phe(1413)-Leu impaired the biosynthetic processing of CFTR, indicating that Phe(1413) is indispensable for the native structure of CFTR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals.
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Program in Cell and Lung Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Howard
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Lukacs
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D017136:Ion Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
D002712:Chlorides; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator