ABCMdb

PMID: 12529365

Silvis MR, Picciano JA, Bertrand C, Weixel K, Bridges RJ, Bradbury NA
A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates.
J Biol Chem. 2003 Mar 28;278(13):11554-60. Epub 2003 Jan 15., 2003-03-28 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Asn287Tyr N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. Login to comment 21 ABCC7 p.Asn287Tyr Recently an individual with mutation N287Y (991A3T) was identified based on a diagnosis of elevated sweat electrolytes (18). Login to comment 22 ABCC7 p.Asn287Tyr Since tyrosine-based signals are important in endocytic targeting, we hypothesized that the N287Y mutation generated a novel additional internalization signal in CFTR (Fig. 1a), leading to reduced cell surface expression of CFTR as a result of increased endocytic activity. Login to comment 23 ABCC7 p.Asn287Tyr Using site-directed mutagenesis in conjunction with morphological, biochemical, and functional assays, we demonstrate that N287Y CFTR generates a novel endocytic sequence enhancing the endocytic rate of CFTR compared with wild type. Login to comment 40 ABCC7 p.Asn287Tyr The N287Y mutation was introduced into the pFRT CFTR wild-type vector using the QuikChange Site-Directed mutagenesis kit (Stratagene). Login to comment 41 ABCC7 p.Asn287Tyr The sequences of the pcDNA5/FRT-CFTR wild type and N287Y were verified prior to use for expression. Login to comment 42 ABCC7 p.Asn287Tyr Cell Lines and Transfections-293 and CHO Flp-InTM (Invitrogen) isogenic cell lines expressing wild-type, ⌬F508, and N287Y CFTR from the same genomic locus were generated according to the manufacturer`s instructions. Login to comment 45 ABCC7 p.Asn287Tyr Confluent monolayers of Madin-Darby canine kidney cells (type II) grown on permeable filter supports were transiently transfected with either wild-type or N287Y CFTR using calcium phosphate. Login to comment 58 ABCC7 p.Asn287Tyr Patch Clamp Analysis-Whole cell and cell-attached patch clamp studies were performed on CHO Flp-In cells expressing wt CFTR or N287Y CFTR. Login to comment 70 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr RESULTS ER Export and Polarity of CFTR Distribution Is Preserved in N287Y CFTR-The cellular distribution of N287Y CFTR was initially examined since intracellular retention of mutant CFTRs by the ER quality control and subsequent failure to mature to a complex-glycosylated form is the most prevalent form of CF. Login to comment 72 ABCC7 p.Asn287Tyr Schematic representation of CFTR and immunofluorescence localization of ⌬F508, wild-type, and N287Y CFTR. Login to comment 75 ABCC7 p.Asn287Tyr The shaded circle shows the N287Y mutated residue within the second intracellular loop (CL-2). Login to comment 78 ABCC7 p.Asn287Tyr b-d, Flp 293 cells were stably transfected with wild-type (b), ⌬F508 (c), or N287Y (d) CFTR or mutant CFTR. Login to comment 80 ABCC7 p.Asn287Tyr type and N287Y CFTR were seen at the cell surface (Fig. 1, b and d). Login to comment 81 ABCC7 p.Asn287Tyr Similar results were obtained for N287Y CFTR stably expressed in CHO cells (data not shown). Login to comment 82 ABCC7 p.Asn287Tyr These results suggest that intracellular retention is unlikely to account for the clinical phenotype associated with N287Y CFTR. Login to comment 83 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr To determine whether the N287Y mutation altered the polarization of CFTR in epithelial cells, we transiently expressed wild-type and N287Y CFTR in Madin-Darby canine kidney cells. Confocal immunofluorescence microscopy demonstrated that wild-type CFTR was polarized to the apical plasma membrane (Fig. 2a) with little or no staining of the basal membrane (Fig. 2e). Login to comment 85 ABCC7 p.Asn287Tyr Similarly N287Y CFTR was also polarized to the apical plasma membrane (Fig. 2b). Login to comment 86 ABCC7 p.Asn287Tyr The ratio of N287Y CFTR in the apical membrane relative to the basolateral membrane was 8.1 Ϯ 3.1 as determined by domain-selective cell surface biotinylation. Login to comment 88 ABCC7 p.Asn287Tyr Steady-state levels and cell surface expression of wild-type and N287Y CFTR. Login to comment 90 ABCC7 p.Asn287Tyr Equal amounts of metabolically labeled 293 cells expressing wild-type or N287Y CFTR were subject to immunoprecipitation using the M3A7 anti-CFTR antibody followed by phosphorimage analysis (top). Login to comment 94 ABCC7 p.Asn287Tyr b, the expression level of wt and N287Y CFTR at the cell surface (biotinylated) and post-ER compartments (complex-glycosylated). Login to comment 96 ABCC7 p.Asn287Tyr c, glycosidase sensitivity of wild-type and N287Y CFTR. Login to comment 101 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr Polarized distribution of wild-type and N287Y CFTR in Madin-Darby canine kidney cells. Confocal micrograph of Madin-Darby canine kidney II cells transiently expressing wild-type and N287Y CFTR. Login to comment 103 ABCC7 p.Asn287Tyr Images show confocal sections taken at the plane of the apical membrane (a and b), plane of the tight junction (c and d), and plane of the basal membrane (e and f) for wild-type (a, c, and e) and N287Y (b, d, and f) CFTR. Login to comment 104 ABCC7 p.Asn287Tyr g, immunoblot of cells expressing wild-type and N287Y CFTR following domain-selective cell surface biotinylation. Login to comment 107 ABCC7 p.Asn287Tyr Thus, abnormalities in the polarized distribution of N287Y CFTR compared with wild-type CFTR cannot account for the disease phenotype associated with this mutation. Login to comment 108 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr N287Y CFTR Shows Altered Cellular Distribution-Quantitative immunoblot analysis of whole cell lysates from isogenic wild-type and N287Y CFTR-expressing cells revealed that steady-state levels of protein expression were identical in each cell line (Fig. 3, a and b). Login to comment 109 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr Moreover the ratio of fully glycosylated mature band C CFTR to immature core-glycosylated band B CFTR was not altered by the N287Y mutation compared with wild-type CFTR (7.2 Ϯ 0.5 and 6.9 Ϯ 0.5, mean Ϯ S.E., n ϭ 4 for wild-type and N287Y CFTR, respectively). Login to comment 110 ABCC7 p.Asn287Tyr In addition, the complex- and core-glycosylated forms of wild-type and N287Y CFTR could be distinguished by their sensitivity to endoglycosidases, which was not different between the two cell lines (Fig. 3c). Login to comment 112 ABCC7 p.Asn287Tyr In both wild-type and N287Y CFTR-expressing cell lines, biotinylated CFTR was detected as a single band of ϳ170 kDa, consistent with the presence of mature fully glycosylated CFTR at the cell surface. Login to comment 113 ABCC7 p.Asn287Tyr However, densitometric analysis revealed that the level of biotinylated N287Y CFTR was only ϳ50% of that for wild-type CFTR (Fig. 3, a and b). Login to comment 114 ABCC7 p.Asn287Tyr The detection of the mature complex-glycosylated form of wild-type and N287Y CFTR in immunoblots and at the cell surface demonstrates that biosynthesis and intracellular transport occurred. Login to comment 115 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr Biosynthesis of N287Y CFTR Is Not Impaired-To better evaluate the efficiency of N287Y CFTR biosynthesis, pulse-chase experiments were performed (Fig. 4, a and b). Login to comment 121 ABCC7 p.Asn287Tyr Biosynthetic maturation, cell surface targeting, and stability of wild-type and N287Y CFTR. Login to comment 128 ABCC7 p.Asn287Tyr Following pulse labeling of wt or N287Y CFTR for 25 min, plasma membrane insertion of the ion channels was determined by biotinylation during a 1-h chase at 37 °C using freshly dissolved sulfo-NHS-SS-biotin (1 mg/ml) every 15 min. Login to comment 131 ABCC7 p.Asn287Tyr d, integrated densities of wt and N287Y CFTR were obtained by phosphorimage analysis and represent mean Ϯ S.E. (n ϭ 3). Login to comment 139 ABCC7 p.Asn287Tyr The apparently normal biosynthesis of mature fully glycosylated N287Y CFTR suggests that targeting to the plasma membrane is largely intact. Login to comment 140 ABCC7 p.Asn287Tyr Wild-type and N287Y CFTR were pulse-labeled with [35 S]methionine, and those molecules that arrived at the cell surface were biotinylated throughout the subsequent chase. Login to comment 142 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr The cell surface targeting efficiency of N287Y CFTR was 91 Ϯ 4% (mean Ϯ S.E., n ϭ 3) of wild-type CFTR, suggesting that biosynthesis and plasma membrane delivery of N287Y CFTR is largely uncompromised. Login to comment 143 ABCC7 p.Asn287Tyr The turnover of complex-glycosylated wild-type and N287Y CFTR was assessed by pulse-chase labeling. Login to comment 145 ABCC7 p.Asn287Tyr The stability of complex-glycosylated N287Y CFTR stably expressed in 293 cells was not significantly different from that observed for wild-type CFTR. Login to comment 146 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr N287Y CFTR Displays Abnormal Endocytic Trafficking- Since biosynthesis and membrane insertion of N287Y CFTR was uncompromised, we hypothesized that the reduction in cell surface N287Y CFTR compared with the wild type was likely due to an increase in endocytic retrieval from the plasma membrane. Login to comment 148 ABCC7 p.Asn287Tyr Direct evidence for enhanced endocytosis of N287Y CFTR was obtained by determining the amount of thiol-resistant biotinylated CFTR with respect to time. Login to comment 149 ABCC7 p.Asn287Tyr The rapid increase in thiol-resistant biotinylated wild-type and N287Y CFTR indicates that both constructs are internalized with high efficiency (Fig. 5). Login to comment 151 ABCC7 p.Asn287Tyr In contrast, the internalization rate of N287Y CFTR was nearly 2-fold faster (12.4 Ϯ 0.4%/min, n ϭ 4), implying that the tyrosine substitution enhanced the endocytic activity of CFTR. Login to comment 152 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr Channel Density, but Not Single Channel Properties, Is Altered in N287Y CFTR-expressing Cells-We have documented abnormalities in the endocytic, but not biosynthetic, traffic of N287Y CFTR that result in a decrease in biotinylatable CFTR at the cell surface. Login to comment 154 ABCC7 p.Asn287Tyr However, it is formally possible that there are also alterations in the biophysical fingerprint of N287Y CFTR compared with wild type. Login to comment 155 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr To confirm that N287Y CFTR is functional at the plasma membrane, electrophysiological recordings were performed in the whole cell and cell-attached patch configurations on CHO cells stably expressing either wild-type or N287Y CFTR. Login to comment 157 ABCC7 p.Asn287Tyr Expression of both wild-type and N287Y CFTR conferred cAMP-stimulated whole cell currents in CHO cells (Fig. 6) with no change in base-line current in the absence of cAMP. Login to comment 158 ABCC7 p.Asn287Tyr The cAMP-stimulated whole cell conductance was 2.6-fold higher in wt CFTR-expressing cells compared with N287Y CFTR-expressing cells. Login to comment 159 ABCC7 p.Asn287Tyr Cell-attached patch studies revealed the single channel properties of the N287Y CFTR were nearly identical to that of wt CFTR. Login to comment 160 ABCC7 p.Asn287Tyr The single channel conductance`s were 9.7 Ϯ 0.35 versus 9.8 Ϯ 0.39 picosiemens and open probabilities were 0.48 Ϯ 0.04 versus 0.46 Ϯ 0.05 for N287Y CFTR and wt CFTR, respectively. Login to comment 161 ABCC7 p.Asn287Tyr These results strongly support the conclusion that the lower whole cell conductance of the N287Y CFTR-expressing cells is the result of a lower density of functional channels in the plasma membrane. Login to comment 162 ABCC7 p.Asn287Tyr DISCUSSION We have demonstrated that the N287Y mutation in CFTR causes clinical disease by dramatically increasing the rate at which CFTR is sequestered from the plasma membrane without altering its channel properties. Login to comment 173 ABCC7 p.Asn287Tyr Internalization efficiency of CFTR is increased by the N287Y mutation. Login to comment 174 ABCC7 p.Asn287Tyr The rate of removal of CFTR from the cell surface was monitored as an increase in biotinylated CFTR resistant to thiol cleavage of the biotin moiety as described under ''Experimental Procedures.`` Cells stably expressing wild-type (filled circle) or N287Y (open circle) CFTR were subjected to cell surface biotinylation at 4 °C, washed, and left to internalize CFTR at 37 °C. Login to comment 179 ABCC7 p.Asn287Tyr The N287Y mutation, a mutation in the second intracellular loop, results in a channel that is biosynthetically and biophysically normal but has greater endocytosis kinetics compared with wild-type CFTR. Login to comment 181 ABCC7 p.Asn287Tyr Similarly targeting of CFTR to the apical plasma membrane domain of polarized epithelial cells is unaffected by the N287Y mutation. Login to comment 182 ABCC7 p.Asn287Tyr In contrast to other mutations in the second intracellular loop, the N287Y mutation has no functional consequence on the gating kinetics of CFTR. Login to comment 184 ABCC7 p.Asn287Tyr The only physiological consequence of the N287Y mutation is therefore to produce a more rapidly endocytosed protein, reducing steady-state levels in the plasma membrane. Login to comment 185 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr The genotype of the initial patient reported to have the N287Y mutation was ⌬F508/N287Y (18). Login to comment 186 ABCC7 p.Asn287Tyr Since little or no cell surface CFTR is produced by the ⌬F508 allele, the only cell surface CFTR protein is produced by the N287Y allele. Login to comment 188 ABCC7 p.Asn287Tyr The most parsimonious interpretation of our data is that the N287Y mutation generates a novel tyrosine-based endocytic motif. Login to comment 191 ABCC7 p.Asn287Tyr Thus it is possible that the N287Y mutation results in the generation of a sequence that displays modest affinity for the AP-2 clathrin adaptor complex. Login to comment 192 ABCC7 p.Asn287Tyr ABCC7 p.Asn287Tyr Although we have focused upon the role of the N287Y mutation in mediating enhanced endocytosis of CFTR, it is still a formal possibility that the N287Y mutation could also reduce the endocytic recycling rate. Login to comment 194 ABCC7 p.Asn287Tyr It is of interest to note that the N287Y mutation is a gain of function mutation and appears to generate an endocytic signal that is present within the body of the protein rather than at the termini of the protein, a localization not previously identified in polytopic membrane proteins. Login to comment