ABCMdb

ABCC7 p.Phe1413Ala

Predicted by SNAP2:	A: D (59%), C: N (66%), D: D (85%), E: D (75%), G: D (75%), H: D (75%), I: N (87%), K: D (80%), L: N (78%), M: D (59%), N: D (75%), P: D (85%), Q: D (71%), R: D (80%), S: D (66%), T: D (71%), V: N (82%), W: D (75%), Y: N (78%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: D, Y: N,

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Publications
[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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Sentences [show]
No. Sentence Comment
40 F1413A/ L1414A/V1415A/I1416A/E1417A, 5Ј-GCTGGAATGCCAACAAGCTGC- GGCCGCAGCAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTC- TCTGCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3Ј; F1413A- /L1414A/V1415A/I1416A, 5Ј-GCTGGAATGCCAACAAGCTGCGGCCG- CAGAAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTCTCTTC- TGCGGCCGCAGCTTGTTGGCATTCCAGC-3Ј; Q1411A/Q1412A, 5Ј-G- GATAGAAGCAATGCTGGAATGCGCAGCATTTTTGGTCATAGAAG-3 and 5Ј-CTTCTATGACCAAAAATGCTGCGCATTCCAGCATTGCTTCT- ATCC-3; F1413A/L1414A, 5Ј-GCTGGAATGCCAACAAGCTGCGGTCA- TAGAAGAGAACAAAGTGCG-3Ј and 5Ј-CGCACTTTGTTCTCTTCTA- TGACCGCAGCTTGTTGGCATTCCAGC-3Ј; L1414A/V1415A, 5Ј-GCT- GGAATGCCAACAATTTGCGGCCATAGAAGAGAACAAAGTGCGG-3Ј and 5Ј-CCGCACTTTGTTCTCTTCTATGGCCGCAAATTGTTGGCATT- CCAGC-3Ј; V1415A/I1416A, 5Ј-GGAATGCCAACAATTTTTGGCCGCA- GAAGAGAACAAAGTGCGGCAG-3Ј and 5Ј-CTGCCGCACTTTGTTCT- CTTCTGCGGCCAAAAATTGTTGGCATTCC-3Ј; E1417A/E1418A, 5Ј- GCCAACAATTTTTGGTCATAGCAGCGAACAAAGTGCGGCAGTAC- G-3Ј and 5Ј-CGTACTGCCGCACTTTGTTCGCTGCTATGACCAAAAA- TTGTTGGC-3Ј; F1413A, 5Ј-GCAATGCTGGAATGCCAACAAGCTTTG- GTCATAGAAGAGAAC-3Ј and 5Ј-GTTCTCTTCTATGACCAAAGCTT- GTTGGCATTCCAGCATTGC-3Ј; L1414A, 5Ј-GCTGGAATGCCAACAA- TTTGCGGTCATAGAAGAGAACAAAGTGCG-3Ј and 5Ј-CGCACTTTG- TTCTCTTCTATGACCGCAAATTGTTGGCATTCCAGC-3Ј; V1415A, 5Ј- GGAATGCCAACAATTTTTGGCCATAGAAGAGAACAAAGTGCGGC- AG-3Ј and 5Ј-CTGCCGCACTTTGTTCTCTTCTATGGCCAAAAATTGT- TGGCATTCC-3Ј; I1416A, 5Ј-GGAATGCCAACAATTTTTGGTCGCAG- AAGAGAACAAAGTGCGGCAG-3Ј and 5Ј-CTGCCGCACTTTGTTCTC- TTCTGCGACCAAAAATTGTTGGCATTCC-3Ј; E1417A, 5Ј-GCCAACA- ATTTTTGGTCATAGCAGAGAACAAAGTGCGGCAGTACG-3Ј and 5Ј- CGTACTGCCGCACTTTGTTCTCTGCTATGACCAAAAATTGTTGGC- 3Ј; C1400A/E1401A/H1402A/R1403A/I1404A, 5Ј-GCACAGTAATTCTC- GCTGCAGCCGCGGCAGAAGCAATGCTGGAATGCC-3Ј and 5Ј-GGC- ATTCCAGCATTGCTTCTGCCGCGGCTGCAGCGAGAATTACTGTG- C-3Ј; ⌬1400-1404: 5Ј-GCATTTGCTGATTGCACAGTAATTCTCGAAG- CAATGCTGGAATGCC-3Ј and 5Ј-GGCATTCCAGCATTGCTTCGAGA- ATTACTGTGCAATCAGCAAATGC-3Ј; C1400A/E1401A, 5Ј-GATTGC- ACAGTAATTCTCGCTGCACACAGGATAGAAGCAATGC-3Ј and 5Ј-G- CATTGCTTCTATCCTGTGTGCAGCGAGAATTACTGTGCAATC-3Ј; H1402A/R1403A, 5Ј-CAGTAATTCTCTGTGAAGCCGCGATAGAAGC- AATGCTGGAATGCC-3Ј and 5Ј-GGCATTCCAGCATTGCTTCTATCG- CGGCTTCACAGAGAATTAC TG-3Ј; I1404A/E1405A, 5Ј-CTCTGTGA- ACACAGGGCAGCAGCAATGCTGGAATGCCAAC-3Ј and 5Ј-GTTGGC- ATTCCAGCATTGCTGCTGCCCTGTGTTCACAGAG-3Ј, Q1390A/A- 1391A/F1392A/A1393A/D1394A, 5Ј-GAAGAACTCTAAAAGCAGCAG- CTGCTGCTTGCACAGTAATTCTC-3Ј and 5Ј-GAGAATTACTGTGCA- AGCAGCAGCTGCTGCTTTTAGAGTTCTTC-3Ј. Login to comment

[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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Sentences [show]
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. Login to comment
171 Considering that the proximal signal might be a combination of an atypical Tyr-based motif (Phe"%"$-Leu-Val-Ile) and a Leu-based motif (Phe"%"$-Leu or Leu"%"%-Val), both Phe"%"$ and Leu"%"% were replaced with alanine in the K2 mutant (F1413A,L1414A) (Figure 5). Login to comment
200 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). Login to comment
269 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). Login to comment