ABCMdb

PMID: 12376531

Peter K, Varga K, Bebok Z, McNicholas-Bevensee CM, Schwiebert L, Sorscher EJ, Schwiebert EM, Collawn JF
Ablation of internalization signals in the carboxyl-terminal tail of the cystic fibrosis transmembrane conductance regulator enhances cell surface expression.
J Biol Chem. 2002 Dec 20;277(51):49952-7. Epub 2002 Oct 9., 2002-12-20 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Tyr1424Ala Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Login to comment 2 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Login to comment 3 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Login to comment 4 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Login to comment 18 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala We find that the substitution of Tyr1424 and Ile1427 with alanine residues resulted in a 2-fold increase in surface expression, whereas the single Y1424A mutation shows an intermediate phenotype. Login to comment 20 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Because the chloride channel activity and relative surface expression of Y1424A and I1427A CFTR are elevated to a similar extent, we propose that these substitutions affect protein trafficking but not CFTR chloride channel function. Login to comment 23 ABCC7 p.Tyr1424Ala The construction of the Y1424A mutant was described previously (3). Login to comment 24 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala For construction of the Y1424A,I1427A mutant, a BstXI-SgrAI fragment that coded for the COOH-terminal tail region of Y1424A CFTR was subcloned into pSK-Bluescript (Stratagene). Login to comment 77 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR were surface-biotinylated and lysed in RIPA buffer (see "Materials and Methods"). Login to comment 82 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The levels of expression of wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were analyzed in COS-7 cells 48 h after transfection. Login to comment 90 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The relative amounts of wild-type (lanes 2 and 6), Y1424A (lanes 3 and 7), and Y1424A,I1427A CFTR (lanes 4 and 8) are shown. Login to comment 94 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The percentage CFTR at the cell surface was markedly increased for Y1424A,I1427A CFTR compared with both wild-type (108% increase, n ϭ 10, p Ͻ 0.001) and Y1424A CFTR (59% increase, n ϭ 10, p Ͻ 0.001) (Fig. 1, bottom panel). Login to comment 97 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Mutations at Tyr1424 and Ile1427 Do Not Alter CFTR Maturation Efficiency or Protein Half-life-To test the effects of these mutations on maturation efficiency and protein half-life, we performed metabolic pulse-chase experiments on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR. Login to comment 99 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The results in Fig. 2 show that the half-lives for wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were 10.3 Ϯ 2.3, 11.3 Ϯ 2.6, and 11.3 Ϯ 1.5 h (mean Ϯ S.D.). Login to comment 102 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The average maturation efficiency for wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were 32, 31, and 31%, respectively (bottom right panel). Login to comment 103 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala This finding demonstrated that elevated surface expression of Y1424A,I1427A CFTR was not because of alterations in maturation efficiency. Login to comment 114 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala COS-7 cells transfected with wild-type, Y1424A, or Y1424A,I1427A CFTR were analyzed 48-h post-transfection. Login to comment 119 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The percentage of wild-type, Y1424A, and Y1424A,I1427A CFTR internalized after 2.5 min was 34, 18, and 8 respectively. Login to comment 122 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala attributed to alterations in the internalization rate of CFTR, we performed internalization assays on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR. Login to comment 128 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala For Y1424A and Y1424A,I1427A CFTR, internalization dropped to 21 and 8%, respectively, during the same time period. Login to comment 130 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala The Y1424A and Y1424A,I1427A CFTR Have Normal Chloride Channel Properties-Because the biochemical data suggested that a specific motif in the CFTR COOH terminus dramatically affected endocytosis and because point mutations in the NH2 terminus lead to both disruption of binding to docking machinery and changes in CFTR ion channel function, we tested whether the mutation of Tyr1424 and Ile1427 affected chloride channel function. Login to comment 136 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala In agreement with the surface biotinylation assays, CFTR whole cell Cl- currents in Y1424A CFTR and Y1424A,I1427A CFTR-transfected cells were elevated compared with wild-type CFTR-expressing cells (Table I), suggesting that the elevated Cl-channel activity was the result of the elevated surface expression of CFTR. Login to comment 140 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Fig. 4, C and D, show the Y1424A and Y1424A,I1427A Cl- current- voltage relationships, respectively, and indicate that although the sensitivities to DIDS and glibenclamide remain similar to wild-type (Fig. 4B), the total current is elevated in the single and double mutants. Login to comment 142 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Single channel biophysical properties of wild-type, Y1424A, and Y1424A,I1427A CFTR were also assessed. Login to comment 145 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Representative recordings of wild-type, Y1424A, and Y1424A,I1427A CFTR at 50-60 mV (negative to pipette potential) are shown in Fig. 4E. Login to comment 150 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala ABCC7 p.Ile1427Ala Nevertheless, the whole cell and single channel recordings together show that the difference in Cl-channel activity is attributed to elevated surface expression without a significant change in CFTR chloride channel properties among wild-type, Y1424A, and Y1424A,I1427A CFTR. Login to comment 154 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala In examining the mechanism for the elevated surface expression of CFTR, we first showed that total expression levels of wild-type, Y1424A, and Y1424A,I1427A were the same. Login to comment 155 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala We next demonstrated that maturation efficiency and protein half-life were unaffected, suggesting that a primary alteration caused by these substitutions involved changes in distribution TABLE I Summary of whole cell patch clamp recordings for wild-type CFTR and for CFTR mutants shows elevated activity in the mutants relative to wild type Transient transfection cAMP-activated chloride currenta Non-green Green Control Wild type Y1424A Y1424A/I1427A pA at ϩ100 mV Set 1 255 Ϯ 36b (13)c 1070 Ϯ 95 (5) 1650 Ϯ 200* (5) 2125 Ϯ 195† (5) (Fold-difference) 1.0 1.54 1.99 Set 2 201 Ϯ 68 (3) 738 Ϯ 52 (5) 986 Ϯ 24* (5) 1451 Ϯ 35† (5) (Fold-difference) 1.0 1.34 1.97 Set 3 393 Ϯ 140 (4) 1193 Ϯ 55 (7) 1767 Ϯ 164* (7) 3424 Ϯ 205† (6) (Fold-difference) 1.0 1.48 2.87 Fold-difference average 1.0 1.45 Ϯ 0.06* 2.28 Ϯ 0.30† a Three sets of transiently transfected COS-7 cells were analyzed in parallel with the protein biochemistry. Login to comment 161 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Moreover, we showed that Y1424A,I1427A CFTR was internalized much more slowly than the native protein (76% inhibition at 2.5 min) with an internalization rate of ϳ2%/min. Login to comment 165 ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Second, the internalization rate of Y1424A,I1427A CFTR is comparable with the rate of bulk flow lipid uptake via the endocytic pathway (ϳ2%/min.) Login to comment 175 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala ABCC7 p.Ile1427Ala Typical whole-cell I-V plots for wild-type CFTR (panel B), Y1424A (panel C), and Y1424A,I1427A (panel D) showing cyclic AMP-stimulated chloride currents in the absence of blockers (squares), presence of DIDS (upward triangles), and presence of glibenclamide (inverted triangles). Login to comment