ABCC7 p.Ala1440*
[switch to full view]Comments [show]
None has been submitted yet.
PMID: 11022033
[PubMed]
Gentzsch M et al: "Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability."
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.).
X
ABCC7 p.Ala1440* 11022033:25:69
status: NEW
PMID: 9920908
[PubMed]
Prince LS et al: "Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal."
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.
X
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).
X
ABCC7 p.Ala1440* 9920908:42:63
status: NEW116 A1440X contains a stop mutation at residue 1440, and Y1424A contains an alanine substitution for tyrosine.
X
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).
X
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).
X
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).
X
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.
X
ABCC7 p.Ala1440* 9920908:133:19
status: NEW137 A, internalization of CFTR and A1440X in COS-7 cells.
X
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.
X
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.
X
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.
X
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.
X
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).
X
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.
X
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).
X
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).
X
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).
X
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).
X
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.
X
ABCC7 p.Ala1440* 9920908:224:80
status: NEW