Fig. 6.

The effect of sorting signal and PDZ-domain-binding motif mutation on CFTR polarized sorting and function in epithelia. (A) Mutagenesis of sorting signals that may influence CFTR transcytosis. Endocytic consensus sites Tyr¹⁴²⁴ (Y) and Leu^1430-1431 (L) were mutated to Ala (A). NN represents Asn⁸⁹⁵ and Asn⁹⁰⁰, two N-linked glycosylation sites in the MSD2. DTRL, PDZ-domain-binding motif at the C-terminus; MSD, membrane spanning domain; NBD, nucleotide-binding domain; R, regulatory domain. (B) Deletion of the DTRL motif (Δ6) increases CFTR apical transcytosis relative to WT-CFTR, but the K3 and N900D mutations do not. (C) Biosynthetic basolateral missorting of CFTR depends on the binding of PDZ-domain protein(s). CFTR apical transcytosis was measured in control (+dox) or dox-OFF (24 h after washout) cells. (D) Apical but not basolateral CFTR internalization is accelerated upon deletion of the PDZ-binding motif (two-tailed unpaired t-test). (E) The Δ6 mutation impairs CFTR apical recycling (n=6), as measured by PM-ELISA. (F) WT- and Δ6-CFTR basolateral cell-surface density was measured by PM-ELISA (two-tailed unpaired t-test). (G) The effect of the Δ6 mutation on CFTR apical transcytosis in the indicated cell line. (H) Δ6-CFTR is predominantly detected at the apical PM in CFBE by indirect immunostaining, performed as in Fig. 1D. Scale bar: 5 µm. (I) The apical PM density and channel function of WT- and Δ6-CFTR were measured by PM-ELISA and by determining the 10 µM forskolin-stimulated short-circuit current (Isc), respectively, and corrected for their relative mRNA level (n=3). The CFTR-mediated Isc was quantified after inhibition with CFTR_inh 172. (J) Apical stability of WT- and Δ6-CFTR is similar. CFTR was labeled with anti-HA (1 h, ice) and chased for the indicated time before PM-ELISA (two-tailed unpaired t-test). Data are mean±s.e.m. on each panel, parentheses indicate the number of independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n.s. not significant.