ABCC7 p.Gly723*
| ClinVar: |
c.2168G>T
,
p.Gly723Val
?
, not provided
|
| CF databases: |
c.2168G>T
,
p.Gly723Val
(CFTR1)
?
,
|
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Comments [show]
None has been submitted yet.
[hide] R-domain interactions with distal regions of CFTR ... Biochemistry. 2000 Aug 15;39(32):9868-75. King SA, Sorscher EJ
R-domain interactions with distal regions of CFTR lead to phosphorylation and activation.
Biochemistry. 2000 Aug 15;39(32):9868-75., 2000-08-15 [PMID:10933805]
Abstract [show]
Cystic fibrosis is caused by the aberrant function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. We examined whether intramolecular binding interactions involving the regulatory (R) domain contributed to CFTR regulation and function. When the R-domain (amino acids 596-836) was coexpressed with Delta1-836 CFTR (a carboxyl hemi-CFTR beginning immediately after the R-domain), strong binding between the two polypeptides was exhibited. The R-domain that co-immunoprecipitated with Delta1-836 exhibited a slower mobility on SDS-PAGE that resulted from phosphorylation of the protein. A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with Delta1-836. Moreover, coexpression of M837X and Delta1-836 led to enhanced halide permeability in living cells. The activity, unlike in full-length CFTR, was present without forskolin activation, but still sensitive to the PKA inhibitor, Rp-8-CPT-cAMPS. This PKA inhibition of activity was found to be dependent on the carboxy region of the R-domain, amino acids 723-836. Our results indicate that the R-domain binds CFTR residues after amino acid 836 and that this binding facilitates phosphorylation and CFTR activation. We have also characterized a subdomain within CFTR (residues 723-837) that is necessary for PKA-dependent constitutive activation. Finally, these experiments demonstrate that constitutive CFTR activity can be accomplished by at least two mechanisms: (1) direct modulation of the R-domain to abrogate PKA regulation and (2) modifications that increase R-domain susceptibility to steady-state phosphorylation through PKA.
Comments [show]
None has been submitted yet.
No. Sentence Comment
27 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP binding cassette; NBD, nucleotide binding domain; TMD, transmembrane domain; R-domain, regulatory domain; PKA, cyclic AMP-dependent protein kinase A; His P, NBD of histidine permease; His Q and His M, TMDs of histidine permease; Mal K, NBD region of the maltose transport system; Mal F, integral membrane protein of the maltose transport system; AMP, adenosine monophosphate; ∆R-CFTR, CFTR lacking amino acids 708-835; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco`s Modified Eagle`s Medium; FBS, fetal bovine serum; vTF7.3, vaccina virus encoding the T7 polymerase; MOI, multiplicity of infection; DOC, deoxycholic acid; PVDF, poly- (vinylidene difluoride); NBT, 4-nitroblue tetrazolium chloride; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolonium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; -gal, -galactosidase; ∆1-836, carboxy hemi-CFTR beginning immediately after the R-domain; M837X, CFTR truncation that ends at CFTR position 836, after the R-domain; G723X, CFTR truncation ending at residue 722 within the R-domain; K593X, CFTR truncation ending immediately before the R-domain at position 592.
X
ABCC7 p.Gly723* 10933805:27:1110
status: NEW53 The resulting product was inserted between the SphI and StuI sites of pTM-CFTR, to place a premature stop codon in place of CFTR amino acid 837. pTM-G723X was constructed using a similar strategy but with a stop codon in place of the glycine at residue 723.
X
ABCC7 p.Gly723* 10933805:53:149
status: NEW173 G723X (missing the 114 C-terminal amino acids of the R-domain, Table 1) was tested for the phosphorylation-dependent mobility shift following coexpression with ∆1-836.
X
ABCC7 p.Gly723* 10933805:173:0
status: NEW174 While G723X co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X, this interaction did not result in a mobility shift of the G723X protein (Figure 7A, compare lanes 3 and 4).
X
ABCC7 p.Gly723* 10933805:174:6
status: NEWX
ABCC7 p.Gly723* 10933805:174:150
status: NEW175 Furthermore, no molecular weight shift of G723X could be detected when cells were treated with forskolin prior to lysis (Figure 7B, lane 4).
X
ABCC7 p.Gly723* 10933805:175:42
status: NEW176 G723X and ∆1-836 expressed together produced constitutive (unregulated) activity (Figure 8A).
X
ABCC7 p.Gly723* 10933805:176:0
status: NEW179 K593X (CFTR truncated immediately before the R-domain, Table 1) co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X or G723X (data not shown), but coexpression of K593X with ∆1-836 failed to produce enhanced halide efflux (Figure 8A).
X
ABCC7 p.Gly723* 10933805:179:145
status: NEW188 A shorter amino truncation (G723X) also tightly bound to ∆1-836.
X
ABCC7 p.Gly723* 10933805:188:28
status: NEW189 In contrast to M837X, G723X failed to exhibit a reduced mobility on SDS-PAGE after either coexpression with ∆1FIGURE 6: Halide permeability produced by M837X and ∆1-836 is PKA-dependent.
X
ABCC7 p.Gly723* 10933805:189:22
status: NEW197 FIGURE 7: G723X binds ∆1-836, but this interaction does not elicit a mobility shift of G723X.
X
ABCC7 p.Gly723* 10933805:197:10
status: NEWX
ABCC7 p.Gly723* 10933805:197:94
status: NEW199 While both G723X and M837X bind ∆1-836 (lanes 3 and 4), only the interaction of M837X with ∆1-836 produced a higher-molecular weight phosphorylated protein (*, lane 3).
X
ABCC7 p.Gly723* 10933805:199:11
status: NEW200 (B) Cells expressing M837X or G723X were treated with forskolin prior to lysis (as described in the legend of Figure 4).
X
ABCC7 p.Gly723* 10933805:200:30
status: NEW202 However, forskolin treament of cells expressing G723X did not result in a higher-molecular weight protein (lane 4).
X
ABCC7 p.Gly723* 10933805:202:48
status: NEW211 G723X also produced high basal halide efflux when it was coexpressed with ∆1-836 (Figure 8A).
X
ABCC7 p.Gly723* 10933805:211:0
status: NEW228 FIGURE 8: G723X coexpression with ∆1-836 produces enhanced halide efflux, while K593X does not.
X
ABCC7 p.Gly723* 10933805:228:10
status: NEW229 (A) G723X and ∆1-836 (9) or K593X and ∆1-836 (b) were expressed in COS7 cells, and halide efflux was assayed as described in the legend of Figure 5.
X
ABCC7 p.Gly723* 10933805:229:4
status: NEW230 G723X produced high basal halide permeability when coexpressed with ∆1-836 (p < 0.01) similar to that of M837X with ∆1-836 (Figure 5).
X
ABCC7 p.Gly723* 10933805:230:0
status: NEW234 (B) M837X and ∆1-836 (0) or G723X and ∆1-836 (O) were expressed in COS7 cells and assayed for halide movement as described in the legend of Figure 6.
X
ABCC7 p.Gly723* 10933805:234:35
status: NEW235 Rp-8-CPT- cAMPS inhibited the enhanced halide permeability of M837X and ∆1-836 (9, p < 0.05), but not that of G723X and ∆1-836 (b).
X
ABCC7 p.Gly723* 10933805:235:117
status: NEW