ABCC7 p.Ala1440*
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[hide] Localization of sequences within the C-terminal do... J Biol Chem. 2001 Jan 12;276(2):1291-8. Gentzsch M, Riordan JR
Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability.
J Biol Chem. 2001 Jan 12;276(2):1291-8., 2001-01-12 [PMID:11022033]
Abstract [show]
Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873-21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413-1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR.
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No. Sentence Comment
25 The antisense primers introducing the stop codon were the following: A1440X, 5Ј-TGATATCGGGC- CCCTATTGCCGGAAGAGGCTCCTCTCGTT-3Ј; S1435X, 5Ј-TGATAT- CGGGCCCCTACCTCTCGTTCAGCAGTTTCTGGATGG-3Ј; L1430X, 5Ј-TGATATCGGGCCCCTATTTCTGGATGGAATCGTACTGCCGCAC- 3Ј; D1425X, 5Ј-TGATATCGGGCCCCTAGTACTGCCGCACTTTGTTC- TCTTCTATG-3Ј; K1420X, 5Ј-TGATATCGGGCCCCTAGTTCTCTTCT- ATGACCAAAAATTGTTGGC-3Ј; V1415X, 5Ј-TGATATCGGGCCCCTA- CAAAAATTGTTGGCATTCCAGCATTGC-3Ј; C1410X, 5Ј-TGATATCG- * This work was supported by National Institutes of Health, NIDDK Grant DK54076 and a Fellowship from the Deutsche Forschungsgemeinschaft (to M. G.).
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ABCC7 p.Ala1440* 11022033:25:69
status: NEW[hide] Efficient endocytosis of the cystic fibrosis trans... J Biol Chem. 1999 Feb 5;274(6):3602-9. Prince LS, Peter K, Hatton SR, Zaliauskiene L, Cotlin LF, Clancy JP, Marchase RB, Collawn JF
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.
J Biol Chem. 1999 Feb 5;274(6):3602-9., 1999-02-05 [PMID:9920908]
Abstract [show]
We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2-78) and carboxyl-terminal tail regions (residues 1391-1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440-1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had approximately 40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.
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No. Sentence Comment
8 We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had ϳ40% reduced internalization activity.
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ABCC7 p.Ala1440* 9920908:8:33
status: NEW42 Construction of CFTR Mutants-pMT-CFTR (wild type) and pMT-CFTR-A1440X were kindly provided by Dr. Seng Cheng (Genzyme) (30).
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ABCC7 p.Ala1440* 9920908:42:63
status: NEW116 A1440X contains a stop mutation at residue 1440, and Y1424A contains an alanine substitution for tyrosine.
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ABCC7 p.Ala1440* 9920908:116:0
status: NEW130 Using a cell surface two-step biotinylation assay to monitor CFTR endocytosis (5) that relies on biotinylation of the carbohydrate side chains found in extracellular loop 4 (Fig. 4), we compared the internalization rate of wild-type CFTR to a previously described premature stop mutant, A1440X (33).
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ABCC7 p.Ala1440* 9920908:130:287
status: NEW131 First, we confirmed that A1440X expressed in COS-7 cells is maturely glycosylated by monitoring band C formation (Fig. 5A).
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ABCC7 p.Ala1440* 9920908:131:25
status: NEW132 Next, using the surface biotinylation assay, we monitored CFTR and A1440X clearance from the cell surface (Fig. 6A).
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ABCC7 p.Ala1440* 9920908:132:67
status: NEW133 Interestingly, the A1440X premature stop mutant was internalized faster than the wild-type CFTR, suggesting, as had been seen for the chimeric protein, that the last 41 residues in CFTR were not necessary for rapid endocytosis.
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ABCC7 p.Ala1440* 9920908:133:19
status: NEW137 A, internalization of CFTR and A1440X in COS-7 cells.
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ABCC7 p.Ala1440* 9920908:137:31
status: NEW138 COS-7 cells transfected with wild-type CFTR (pMT-CFTR) or A1440X (pMT-1440X) were analyzed 48 h posttransfection.
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ABCC7 p.Ala1440* 9920908:138:58
status: NEW142 Biotinylated and nonbiotinylated proteins were separated on a monomeric avidin column, and CFTR and A1440X were in vitro phosphorylated and analyzed by SDS-polyacrylamide gels and autoradiography to quantitate the amount of CFTR remaining on the cell surface during the warm-up step.
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ABCC7 p.Ala1440* 9920908:142:100
status: NEW143 Each time point represents the mean Ϯ S.E. of 13 experiments for wild-type CFTR and 6 for the A1440X.
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ABCC7 p.Ala1440* 9920908:143:100
status: NEW168 As is shown in Fig. 7, the wild-type CFTR protein and A1440X (Fig. 7, A and B, respectively) showed a strong juxtanuclear distribution and evidence of surface staining (a clear outline of the cell borders), whereas all of the deletion mutants had a reticular staining pattern and little if any evidence of surface staining.
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ABCC7 p.Ala1440* 9920908:168:54
status: NEW173 The A1440X mutant, unlike the amino-terminal deletion mutants, had wild-type chloride channel activity (Fig. 8B).
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ABCC7 p.Ala1440* 9920908:173:4
status: NEW183 COS-7 cells transfected with wild-type CFTR (A), A1440X (B), ⌬2-79 CFTR (C), ⌬2-59 CFTR (D), ⌬35-45 CFTR (E), and ⌬60-72 (F) were fixed, permeabilized, and subjected to indirect immunofluorescence using anti-CFTR antibodies.
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ABCC7 p.Ala1440* 9920908:183:49
status: NEW184 Wild-type CFTR and A1440X show cell surface staining, whereas the amino-terminal deletion mutants show a pronounced reticular staining pattern. internalization, it is tempting to speculate that the CFTR protein contains more than one internalization signal, such as is the case for the CD3 ␥-chain (13).
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ABCC7 p.Ala1440* 9920908:184:19
status: NEW211 The change in SPQ fluorescence is shown for COS-7 cells expressing wild-type CFTR, ⌬2-79 CFTR, ⌬2-59 CFTR, ⌬35-45 CFTR, and ⌬60-72 CFTR (A) and wild-type CFTR and A1440X (B).
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ABCC7 p.Ala1440* 9920908:211:191
status: NEW214 Curves were generated from either 1) the mean Ϯ S.E. of responding cells (defined by increased rate of dequench following cAMP stimulation of Ͼ100% (wild type CFTR (A), wild type CFTR and A1440X CFTR (B)) or 2) the total of all screened cells expressing a given construct. The values in parentheses are the number of responding cells (numerator) over the total cells screened (denominator).
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ABCC7 p.Ala1440* 9920908:214:200
status: NEW216 B, the wild type CFTR and A1440X CFTR samples were significantly different from the mock cells (p Ͻ 0.001 and 0.025, respectively).
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ABCC7 p.Ala1440* 9920908:216:26
status: NEW224 Clearly, a direct comparison of the internalization rates of wild-type CFTR and A1440X mutant in a polarized epithelial cell line will be required to clarify the role of the carboxyl terminus in membrane association and/or endocytosis.
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ABCC7 p.Ala1440* 9920908:224:80
status: NEW