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 Tyr1424-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr1424 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 α 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 (Phe1413) and a bipartite endocytic signal, comprising a tyrosine (Tyr1424) and a di-Leu-based (Leu1430-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 Phe1413-Leu impaired the biosynthetic processing of CFTR, indicating that Phe1413 is indispensable for the native structure of CFTR. In contrast, replacement of Leu1430-Leu- and Tyr1424-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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