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-de pendent protein kinase (PKA)-activated chloride channel that is localized t o the plasma membrane and endosomal compartment, Endosomal targeting of CFT R is attributed to the Tyr(1424)-based internalization signal, identified i n the C-terminal tail of the channel. Mutation of the Tyr(1424) residue cou ld partly inhibit the endocytosis of CFTR and its association with the adap ter protein AP-2. To reveal additional endosomal targeting signals, site-di rected mutagenesis of both a chimaera, composed of a truncated form of inte rleukin 2 receptor alpha chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assay s revealed the presence of multiple internalization motifs at the C-terminu s, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite en docytic signal, comprising a tyrosine (Tyr(1424)) and a di-leu-based (Leu(1 430)-Leu) motif. Whereas the replacement of any one of the three internaliz ation 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 CF TR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and C FTR in an additive manner. These results suggest that the internalization o f CFTR is regulated by multiple endocytic sorting signals.