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.
Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes
