ABCC7 p.Ile1427Ala
| ClinVar: |
c.4280T>C
,
p.Ile1427Thr
D
, Likely pathogenic
|
| Predicted by SNAP2: | A: N (93%), C: N (87%), D: D (66%), E: D (59%), F: N (87%), G: D (53%), H: N (57%), K: N (53%), L: N (97%), M: N (97%), N: N (53%), P: N (61%), Q: N (53%), R: D (53%), S: N (61%), T: N (93%), V: N (93%), W: D (53%), Y: N (57%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Ablation of internalization signals in the carboxy... J Biol Chem. 2002 Dec 20;277(51):49952-7. Epub 2002 Oct 9. 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 [PMID:12376531]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. 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). 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. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. 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. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.
Comments [show]
None has been submitted yet.
No. Sentence Comment
2 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.
X
ABCC7 p.Ile1427Ala 12376531:2:79
status: NEW3 Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis.
X
ABCC7 p.Ile1427Ala 12376531:3:75
status: NEW4 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.
X
ABCC7 p.Ile1427Ala 12376531:4:139
status: NEW18 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.
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ABCC7 p.Ile1427Ala 12376531:18:45
status: NEW20 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.
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ABCC7 p.Ile1427Ala 12376531:20:84
status: NEW24 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).
X
ABCC7 p.Ile1427Ala 12376531:24:31
status: NEW77 COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR were surface-biotinylated and lysed in RIPA buffer (see "Materials and Methods").
X
ABCC7 p.Ile1427Ala 12376531:77:53
status: NEW82 The levels of expression of wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were analyzed in COS-7 cells 48 h after transfection.
X
ABCC7 p.Ile1427Ala 12376531:82:63
status: NEW90 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.
X
ABCC7 p.Ile1427Ala 12376531:90:86
status: NEW94 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).
X
ABCC7 p.Ile1427Ala 12376531:94:74
status: NEW97 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.
X
ABCC7 p.Ile1427Ala 12376531:97:284
status: NEW99 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.).
X
ABCC7 p.Ile1427Ala 12376531:99:86
status: NEW102 The average maturation efficiency for wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were 32, 31, and 31%, respectively (bottom right panel).
X
ABCC7 p.Ile1427Ala 12376531:102:73
status: NEW103 This finding demonstrated that elevated surface expression of Y1424A,I1427A CFTR was not because of alterations in maturation efficiency.
X
ABCC7 p.Ile1427Ala 12376531:103:69
status: NEW114 COS-7 cells transfected with wild-type, Y1424A, or Y1424A,I1427A CFTR were analyzed 48-h post-transfection.
X
ABCC7 p.Ile1427Ala 12376531:114:58
status: NEW119 The percentage of wild-type, Y1424A, and Y1424A,I1427A CFTR internalized after 2.5 min was 34, 18, and 8 respectively.
X
ABCC7 p.Ile1427Ala 12376531:119:48
status: NEW122 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.
X
ABCC7 p.Ile1427Ala 12376531:122:155
status: NEW128 For Y1424A and Y1424A,I1427A CFTR, internalization dropped to 21 and 8%, respectively, during the same time period.
X
ABCC7 p.Ile1427Ala 12376531:128:22
status: NEW130 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.
X
ABCC7 p.Ile1427Ala 12376531:130:22
status: NEW136 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.
X
ABCC7 p.Ile1427Ala 12376531:136:107
status: NEW140 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.
X
ABCC7 p.Ile1427Ala 12376531:140:44
status: NEW142 Single channel biophysical properties of wild-type, Y1424A, and Y1424A,I1427A CFTR were also assessed.
X
ABCC7 p.Ile1427Ala 12376531:142:71
status: NEW145 Representative recordings of wild-type, Y1424A, and Y1424A,I1427A CFTR at 50-60 mV (negative to pipette potential) are shown in Fig. 4E.
X
ABCC7 p.Ile1427Ala 12376531:145:59
status: NEW150 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.
X
ABCC7 p.Ile1427Ala 12376531:150:262
status: NEW154 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.
X
ABCC7 p.Ile1427Ala 12376531:154:150
status: NEW155 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.
X
ABCC7 p.Ile1427Ala 12376531:155:445
status: NEW161 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.
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ABCC7 p.Ile1427Ala 12376531:161:32
status: NEW165 Second, the internalization rate of Y1424A,I1427A CFTR is comparable with the rate of bulk flow lipid uptake via the endocytic pathway (ϳ2%/min.)
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ABCC7 p.Ile1427Ala 12376531:165:43
status: NEW175 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).
X
ABCC7 p.Ile1427Ala 12376531:175:88
status: NEW