ABCC7 p.Leu1430Ala
| Predicted by SNAP2: | A: D (66%), C: D (59%), D: D (75%), E: D (66%), F: D (59%), G: D (75%), H: D (66%), I: D (53%), K: D (80%), M: N (61%), N: D (71%), P: D (80%), Q: D (63%), R: D (75%), S: D (71%), T: D (71%), V: N (53%), W: D (71%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] Multiple endocytic signals in the C-terminal tail ... Biochem J. 2001 Mar 15;354(Pt 3):561-72. Hu W, Howard M, Lukacs GL
Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator.
Biochem J. 2001 Mar 15;354(Pt 3):561-72., 2001-03-15 [PMID:11237860]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-activated chloride channel that is localized to the plasma membrane and endosomal compartment. Endosomal targeting of CFTR is attributed to the Tyr(1424)-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr(1424) residue could partly inhibit the endocytosis of CFTR and its association with the adapter protein AP-2. To reveal additional endosomal targeting signals, site-directed mutagenesis of both a chimaera, composed of a truncated form of interleukin 2 receptor alpha chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assays revealed the presence of multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite endocytic signal, comprising a tyrosine (Tyr(1424)) and a di-Leu-based (Leu(1430)-Leu) motif. Whereas the replacement of any one of the three internalization motifs with alanine prevented the endocytosis of the TacT-Ct chimaera, mutagenesis of Phe(1413)-Leu impaired the biosynthetic processing of CFTR, indicating that Phe(1413) is indispensable for the native structure of CFTR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals.
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No. Sentence Comment
75 Alanine substitutions in the following mutants were generated by site-directed mutagenesis: K2, F1413A,L1414A; K3, Y1424A,L1430A,L1431A; K6, F1413A; K7, L1414A; K8, Y1424A; K9, L1430A; K10, F16A,F17A (see also Figure 5), by using either single-stranded (for K2, K3, K8 and K9; Muta Gene in itro mutagenesis from Bio-Rad) or double-stranded (for K10; QuickChange site-directed mutagenesis from Stratagene [35]) pcDNA3-TacT-Ct as template.
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ABCC7 p.Leu1430Ala 11237860:75:122
status: NEWX
ABCC7 p.Leu1430Ala 11237860:75:177
status: NEW172 The membrane-distal signals, consisting of a canonical Tyr-based motif and a di-Leu motif, were mutated together and designated as mutant K3 (Y1424A,L1430A,L1431A) (Figure 5).
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ABCC7 p.Leu1430Ala 11237860:172:149
status: NEW200 To evaluate the involvement of single amino acid residues in the membrane-proximal and membrane-distal internalization signals, four additional constructs were prepared that disrupted the three putative endocytic signals individually, as follows: K6, F1413A; K7, L1414A; K8, Y1424A; K9, L1430A (see also Figure 5).
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ABCC7 p.Leu1430Ala 11237860:200:287
status: NEW231 Mutation of the membrane-distal internalization signals (K8, Y1424A; K9, L1430A) did not interfere with the processing of CFTRM2, as demonstrated by the large-amplitude cAMP-dependent iodide release from cells expressing K3CFTRM2, K8CFTRM2 or K9CFTRM2 (Figure 8A).
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ABCC7 p.Leu1430Ala 11237860:231:73
status: NEW269 Alanine substitutions of individual residues in the membrane-proximal (K6, F1413A; K7, L1414A) or the membrane-distal (K8, Y1424A; K9, L1430A) stretch revealed an additive effect on the internalization activity (Figure 7D).
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ABCC7 p.Leu1430Ala 11237860:269:135
status: NEW279 In contrast, the disruption of individual components of the bipartite membrane-distal signal caused a 4-fold (K8, Y1424A) and a 3-fold (K9, L1430A) increase in the cell-surface density of the CFTRM2 (Figure 8B; see also Figure 7D).
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ABCC7 p.Leu1430Ala 11237860:279:140
status: NEW[hide] Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18. Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PMID:19095655]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.
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No. Sentence Comment
43 EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1.
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ABCC7 p.Leu1430Ala 19095655:43:164
status: NEW83 To exclude a nonspecific effect of compound C, we expressed a CFTR mutant (L1430A/ L1431A), which has been proposed to eliminate binding of AMPK␣1 to a C-terminal region of CFTR (5).
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ABCC7 p.Leu1430Ala 19095655:83:75
status: NEW86 This enhanced activity of L1430A/L1431A-CFTR was similar to that seen with wild-type CFTR first exposed to compound C and then activated by PKA.
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ABCC7 p.Leu1430Ala 19095655:86:26
status: NEW103 D, summary of whole cell conductances generated by wtCFTR and L1430A/L1431A-CFTR and effects of activators and inhibitors of AMPK.
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ABCC7 p.Leu1430Ala 19095655:103:62
status: NEW104 E, whole cell currents activated by IBMX and forskolin in wtCFTR and L1430A/L1431A-CFTR expressing oocytes.
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ABCC7 p.Leu1430Ala 19095655:104:69
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
103 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).
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ABCC7 p.Leu1430Ala 9920908:103:161
status: NEW