ABCC7 p.Thr421Ala
Predicted by SNAP2: | A: N (87%), C: N (87%), D: N (78%), E: N (78%), F: N (87%), G: N (93%), H: N (93%), I: N (93%), K: N (82%), L: N (93%), M: N (93%), N: N (93%), P: N (82%), Q: N (93%), R: N (82%), S: N (93%), V: N (93%), W: N (72%), Y: N (97%), |
Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: N, Y: N, |
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[hide] Activation mechanisms for the cystic fibrosis tran... Biochem J. 2007 Jul 1;405(1):181-9. Pereira MM, Parker J, Stratford FL, McPherson M, Dormer RL
Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP.
Biochem J. 2007 Jul 1;405(1):181-9., 2007-07-01 [PMID:17381427]
Abstract [show]
The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1-R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1-R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 microM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 microM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 microM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective DeltaF508-CFTR trafficking.
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No. Sentence Comment
7 Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 µM.
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ABCC7 p.Thr421Ala 17381427:7:28
status: NEW39 The T421A mutation was introduced into the pRSETB vector using Stratagene`s QuikChange® II Site-directed Mutagenesis kit and oligonucleotide primers (forward and reverse) containing the mutation amidst a 21 base-pair 5 - and 3 - flanking region.
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ABCC7 p.Thr421Ala 17381427:39:4
status: NEW154 However, as for ATP binding, cAMP displacement followed a biexponential curve, yielding values for half-maximal displacement of [3 H]cAMP of 2.6 and 167 µM. Sullivan et al. [18] compared the whole-cell chloride channel activity elicited by injection of 50 µM cAMP into cells expressing wild-type CFTR or the mutant form, T421A.
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ABCC7 p.Thr421Ala 17381427:154:331
status: NEW157 We introduced the T421A mutation into CFTR cDNA (see the Experimental section) and expressed an NBD1-R domain protein to investigate the effect on cAMP binding.
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ABCC7 p.Thr421Ala 17381427:157:18
status: NEW158 As shown in Figure 5(B), the T421A mutant protein bound [3 H]cAMP but showed half-maximal displacement by unlabelled cAMP at 9.2 and 201 µM.
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ABCC7 p.Thr421Ala 17381427:158:29
status: NEW159 Thus the results suggest that the T421A mutation results in decreased affinity of the higher affinity binding, which is responsible for activation of CFTR.
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ABCC7 p.Thr421Ala 17381427:159:34
status: NEW177 All the results are means + - S.E.M.; (A) n = 7 for displacement by cAMP; (B) displacement by cAMP from wild-type (closed circles, n = 5) or T421A (open squares, n = 5) NBD1-R domain protein.
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ABCC7 p.Thr421Ala 17381427:177:141
status: NEW218 We synthesized a T421A mutant form of the NBD1-R domain protein and showed a decreased affinity for cAMP binding (Figure 5B), consistent with the demonstration that the full-length T421A CFTR showed reduced chloride channel activity in whole cells [18].
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ABCC7 p.Thr421Ala 17381427:218:17
status: NEWX
ABCC7 p.Thr421Ala 17381427:218:181
status: NEW[hide] Identification and partial characterization of a d... Curr Biol. 1995 Oct 1;5(10):1159-67. Sullivan SK, Agellon LB, Schick R
Identification and partial characterization of a domain in CFTR that may bind cyclic nucleotides directly.
Curr Biol. 1995 Oct 1;5(10):1159-67., [PMID:8548288]
Abstract [show]
BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C. RESULTS: Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87% of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain--Val397 and Lys420--were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP. CONCLUSIONS: We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.
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No. Sentence Comment
125 The current was only 18 + 13 % (n = 3) of wild-type level for the mutant channel with the glutamate at position 407 substituted by glutamine (mutant E407Q, using the single-letter amino-acid code); 16 + 3 % (n = 4) for the L408A mutant; and 17 ±+1 % (n = 4) for the T421A mutant (data not shown).
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ABCC7 p.Thr421Ala 8548288:125:271
status: NEW124 The current was only 18 + 13 % (n = 3) of wild-type level for the mutant channel with the glutamate at position 407 substituted by glutamine (mutant E407Q, using the single-letter amino-acid code); 16 + 3 % (n = 4) for the L408A mutant; and 17 &#b1;+1 % (n = 4) for the T421A mutant (data not shown).
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ABCC7 p.Thr421Ala 8548288:124:270
status: NEW