ABCMdb

ABCC7 p.Leu1431Ala

Predicted by SNAP2:	A: D (63%), C: N (53%), D: D (80%), E: D (75%), F: N (53%), G: D (75%), H: D (71%), I: N (57%), K: D (75%), M: N (78%), N: D (71%), P: D (80%), Q: D (66%), R: D (75%), S: D (66%), T: D (63%), V: N (66%), W: D (66%), Y: D (59%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, 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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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
172 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). Login to comment

[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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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. Login to comment
83 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). Login to comment
86 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. Login to comment
103 D, summary of whole cell conductances generated by wtCFTR and L1430A/L1431A-CFTR and effects of activators and inhibitors of AMPK. Login to comment
104 E, whole cell currents activated by IBMX and forskolin in wtCFTR and L1430A/L1431A-CFTR expressing oocytes. Login to comment