ABCMdb

PMID: 9920908

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 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Tyr1424Ala Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Login to comment 8 ABCC7 p.Tyr1424Ala ABCC7 p.Ala1440* 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. Login to comment 42 ABCC7 p.Ala1440* Construction of CFTR Mutants-pMT-CFTR (wild type) and pMT-CFTR-A1440X were kindly provided by Dr. Seng Cheng (Genzyme) (30). Login to comment 103 ABCC7 p.Tyr1424Ala ABCC7 p.Leu1430Ala ABCC7 p.Tyr38Ala ABCC7 p.Leu69Ala Next, we analyzed CFTR-TR chimeras that contained point mutations in potential internalization signals in both cytoplasmic tail regions: Y38A, L69A, Y1424A, and L1430A (Fig. 1). Login to comment 104 ABCC7 p.Tyr1424Ala Analysis of these mutants in internalization assays indicated that only one mutation, Y1424A, affected the internalization rate of the chimeras (Fig. 2B; ϳ40% loss of internalization activity, p Ͻ 0.05), suggesting that this residue might be a part of an internalization signal. Login to comment 116 ABCC7 p.Tyr1424Ala ABCC7 p.Ala1440* A1440X contains a stop mutation at residue 1440, and Y1424A contains an alanine substitution for tyrosine. Login to comment 123 ABCC7 p.Tyr1424Ala Comparison of the CFTR-(1391-1440) with the Y1424A mutant showed little or no difference (not shown). Login to comment 130 ABCC7 p.Ala1440* 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). Login to comment 131 ABCC7 p.Ala1440* First, we confirmed that A1440X expressed in COS-7 cells is maturely glycosylated by monitoring band C formation (Fig. 5A). Login to comment 132 ABCC7 p.Ala1440* Next, using the surface biotinylation assay, we monitored CFTR and A1440X clearance from the cell surface (Fig. 6A). Login to comment 133 ABCC7 p.Ala1440* 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. Login to comment 134 ABCC7 p.Tyr1424Ala Tyrosine 1424 Is Important for CFTR Endocytosis-Next, we tested the only point mutation that affected internalization of the chimeras, Y1424A. Login to comment 137 ABCC7 p.Ala1440* A, internalization of CFTR and A1440X in COS-7 cells. Login to comment 138 ABCC7 p.Ala1440* COS-7 cells transfected with wild-type CFTR (pMT-CFTR) or A1440X (pMT-1440X) were analyzed 48 h posttransfection. Login to comment 142 ABCC7 p.Ala1440* 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. Login to comment 143 ABCC7 p.Ala1440* Each time point represents the mean Ϯ S.E. of 13 experiments for wild-type CFTR and 6 for the A1440X. Login to comment 144 ABCC7 p.Tyr1424Ala B, internalization of CFTR and Y1424A in COS-7 cells. Login to comment 145 ABCC7 p.Tyr1424Ala Cells transfected with wild-type CFTR (pGT-CFTR) or Y1424A were analyzed as described in A for percentage CFTR internalized from the cell surface during the warm-up period. Login to comment 146 ABCC7 p.Tyr1424Ala C, percentage of CFTR or Y1424A at the cell surface under steady-state conditions. Login to comment 147 ABCC7 p.Tyr1424Ala Cells transfected with CFTR or Y1424A were analyzed for total CFTR expression by performing the two-step biotinylation reaction without a warm-up step. Login to comment 148 ABCC7 p.Tyr1424Ala Biotinylated and nonbiotinylated CFTR or Y1424A was separated on a monovalent avidin column and quantitated as described for A. Login to comment 156 ABCC7 p.Tyr1424Ala tion would imply that the steady-state distribution of Y1424A might favor a higher cell surface distribution pattern, we determined the percentage of CFTR at the cell surface using surface biotinylation at 4 °C. Login to comment 157 ABCC7 p.Tyr1424Ala As predicted, the percentage of Y1424A at the cell surface was higher than the wild-type CFTR protein (36.1 versus 23.1% (p Ͻ 0.01), respectively; Fig. 6C), supporting the idea that tyrosine 1424 was important for CFTR endocytosis. Login to comment 163 ABCC7 p.Tyr1424Ala A similar comparison between wild type CFTR (pGT) and Y1424A (pGT) revealed that the expression levels were similar (Fig. 5B). Login to comment 168 ABCC7 p.Ala1440* 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. Login to comment 173 ABCC7 p.Ala1440* The A1440X mutant, unlike the amino-terminal deletion mutants, had wild-type chloride channel activity (Fig. 8B). Login to comment 178 ABCC7 p.Tyr1424Ala The only mutation that we identified in our studies that affected CFTR internalization was found in the carboxyl-terminal tail, Y1424A. Login to comment 183 ABCC7 p.Ala1440* 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. Login to comment 184 ABCC7 p.Ala1440* 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). Login to comment 196 ABCC7 p.Val1397Glu For comparison, only one mutation in the carboxyl-terminal tail has been reported to result in CF, V1397E. Login to comment 211 ABCC7 p.Ala1440* 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). Login to comment 214 ABCC7 p.Ala1440* 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). Login to comment 216 ABCC7 p.Ala1440* B, the wild type CFTR and A1440X CFTR samples were significantly different from the mock cells (p Ͻ 0.001 and 0.025, respectively). Login to comment 224 ABCC7 p.Ala1440* 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. Login to comment