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PMID: 10933805
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
[PubMed]
Sentences
No.
Mutations
Sentence
Comment
4
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:4:54
status:
NEW
view ABCC7 p.Met837* details
A larger CFTR polypeptide that included the R-domain (
M837X
) also exhibited a phosphorylation-dependent mobility shift when coexpressed with ∆1-836.
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5
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:5:26
status:
NEW
view ABCC7 p.Met837* details
Moreover, coexpression of
M837X
and ∆1-836 led to enhanced halide permeability in living cells.
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27
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:27:1176
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:27:1177
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:27:1035
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:27:1036
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:27:1110
status:
NEW
view ABCC7 p.Gly723* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:27:1111
status:
NEW
view ABCC7 p.Gly723* details
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.
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52
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:52:4
status:
NEW
view ABCC7 p.Met837* details
pTM-
M837X
was produced by amplifying a segment of CFTR with a primer encoding nucleotides 1562-1585 (including the unique SphI restriction site) and a reverse primer containing nucleotides 2495-2529 (including a stop codon at residue 2515 followed by a StuI site).
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53
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:53:149
status:
NEW
view ABCC7 p.Gly723* details
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.
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54
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:54:120
status:
NEW
view ABCC7 p.Met837* details
The pTM-R-domain was obtained using a PCR product to engineer a start site at methionine 596 and the stop site from pTM-
M837X
(leading to a vector expressing the R-domain protein, i.e., amino acids 596-837).
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111
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:111:39
status:
NEW
view ABCC7 p.Met837* details
Interactions between ∆1-836 and
M837X
Confer Phosphorylation of the R-Domain and ActiVation of Halide Efflux.
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112
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:112:170
status:
NEW
view ABCC7 p.Met837* details
To ascertain whether phosphorylation of the R-domain is enhanced by expression of the complete CFTR amino acid sequence, we transfected cells with both ∆1-836 and
M837X
(CFTR truncated at the end of the R-domain, Table 1).
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115
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:115:46
status:
NEW
view ABCC7 p.Met837* details
Immunoprecipitates from COS7 cells expressing
M837X
, ∆1-836, or both were studied by Western blotting, as shown in Figure 3.
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116
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:116:4
status:
NEW
view ABCC7 p.Met837* details
The
M837X
protein was pulled down in association with ∆1-836 (Figure 3A).
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117
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:117:111
status:
NEW
view ABCC7 p.Met837* details
The complementary experiment showed that ∆1-836 could also be co-immunoprecipitated in association with
M837X
(Figure 3B, lane 6).
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118
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:118:8
status:
NEW
view ABCC7 p.Met837* details
Neither
M837X
nor ∆1-836 bound to a control ( -gal) protein (Figure 3C).
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119
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:119:19
status:
NEW
view ABCC7 p.Met837* details
Interestingly, the
M837X
protein that co-immunoprecipitated with ∆1-836 was greatly enriched for an apparent higher-molecular weight conformation (Figure 3A, lane 4 with *).
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120
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:120:30
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:120:78
status:
NEW
view ABCC7 p.Met837* details
Direct immunoprecipitation of
M837X
indicated that the total level of shifted
M837X
protein was negligible when expressed alone (Figure 3A, lane 1).
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121
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:121:5
status:
NEW
view ABCC7 p.Met837* details
When
M837X
was coexpressed with ∆1-836, the higher protein band could be more readily detected, and was dramatically enriched bound to ∆1-836 (Figure 3A, lane 4).
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122
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:122:74
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:122:98
status:
NEW
view ABCC7 p.Met837* details
To test whether phosphorylation was responsible for the mobility shift in
M837X
, cells expressing
M837X
alone were treated with forskolin prior to lysis.
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123
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:123:72
status:
NEW
view ABCC7 p.Met837* details
Forskolin treatment reproduced the slower molecular weight migration in
M837X
(Figure 4A, lane 1).
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124
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:124:53
status:
NEW
view ABCC7 p.Met837* details
Alkaline phosphatase treatment of immunoprecipitated
M837X
protein abolished the higher-molecular weight band seen following either PKA stimulation or coexpression with ∆1-836 (Figure 4A, lanes 2 and 4).
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125
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:125:65
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:125:136
status:
NEW
view ABCC7 p.Met837* details
These results indicate that the expression of ∆1-836 with
M837X
confers a phosphorylation-dependent reduction in the mobility of
M837X
.
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126
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:126:83
status:
NEW
view ABCC7 p.Met837* details
Forskolin treatment did not cause phosphorylation of all the expressed R-domain or
M837X
.
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129
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:129:55
status:
NEW
view ABCC7 p.Met837* details
If this were so, only a subfraction of the R-domain or
M837X
might be situated within the cell in a position suitable for efficient phosphorylation by PKA.
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132
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:132:39
status:
NEW
view ABCC7 p.Met837* details
Since we observed that binding between
M837X
and ∆1-836 augments CFTR phosphorylation, we tested the functional consequences of this binding interaction.
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133
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:133:0
status:
NEW
view ABCC7 p.Met837* details
M837X
, ∆1-836, or both were expressed in COS7 cells, and anion efflux was measured using the halide sensitive dye SPQ.
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134
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:134:16
status:
NEW
view ABCC7 p.Met837* details
Coexpression of
M837X
and ∆1-836 led to augmented cellular halide permeability (Figure 5, b), while no halide efflux above FIGURE 2: Forskolin treatment or coexpression with ∆1-836 produces an apparent higher-molecular weight R-domain protein that is sensitive to alkaline phosphatase.
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141
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:141:51
status:
NEW
view ABCC7 p.Met837* details
background was detected in cells expressing either
M837X
or ∆1-836 alone (Figure 5, 0 and O).
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142
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:142:16
status:
NEW
view ABCC7 p.Met837* details
Coexpression of
M837X
and ∆1-836 produced predominantly constitutive function of the reconstituted CFTR, similar to that reported previously for CFTR lacking a portion of the R-domain (∆R-CFTR) (15, 19).
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146
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:146:41
status:
NEW
view ABCC7 p.Met837* details
The increased halide efflux conferred by
M837X
and ∆1-836 could be either due to the loss of PKA-dependent regulation of CFTR [as demonstrated for ∆R-CFTR (15)] or a result of enhanced R-domain basal PKA phosphorylation.
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148
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:148:24
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:148:204
status:
NEW
view ABCC7 p.Met837* details
When cells coexpressing
M837X
and ∆1-836 were treated with Rp-8-CPT- cAMPS, a PKA antagonist, a significant decrease in the halide efflux was detected compared to that in untreated cells FIGURE 3:
M837X
binds ∆1-836.
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149
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:149:0
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:149:84
status:
NEW
view ABCC7 p.Met837* details
M837X
, ∆1-836, or both were immunoprecipitated using an antibody specific to
M837X
, or to the C-terminal half.
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150
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:150:0
status:
NEW
view ABCC7 p.Met837* details
M837X
co-immunoprecipitated with ∆1-836 (panel A, lane 4).
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151
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:151:74
status:
NEW
view ABCC7 p.Met837* details
The ∆1-836 was also detected following co-immunoprecipitation with
M837X
(panel B, lane 6).
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153
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:153:50
status:
NEW
view ABCC7 p.Met837* details
The asterisk denotes the shifted mobility form of
M837X
; see the text. The antibody used in Western blotting is defined at the right of each panel.
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154
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:154:150
status:
NEW
view ABCC7 p.Met837* details
FIGURE 4: Forskolin treatment or coexpression with ∆1-836 produces an apparent higher-molecular weight, alkaline phosphatase sensitive form of
M837X
.
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155
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:155:30
status:
NEW
view ABCC7 p.Met837* details
Lysates from cells expressing
M837X
, alone or together with ∆1-836 ("both"), were immunoprecipitated as described in the legend of Figure 3.
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157
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:157:0
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:157:44
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:157:129
status:
NEW
view ABCC7 p.Met837* details
M837X
from cells treated with forskolin and
M837X
coexpressed with ∆1-836 resulted in an apparent higher-molecular weight
M837X
-CFTR protein band (*, lanes 1 and 3).
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158
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:158:28
status:
NEW
view ABCC7 p.Met837* details
The higher-molecular weight
M837X
protein band was eliminated by treatment with alkaline phosphatase (lanes 2 and 4).
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159
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:159:10
status:
NEW
view ABCC7 p.Met837* details
FIGURE 5:
M837X
and ∆1-836 coexpression produces elevated basal halide efflux.
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160
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:160:10
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:160:43
status:
NEW
view ABCC7 p.Met837* details
CFTR (9),
M837X
(0), ∆1-836 (O), or
M837X
and ∆1-836 (b) were expressed in COS7 cells, and halide efflux was assayed using the halide sensitive intracellular dye SPQ.
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163
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:163:108
status:
NEW
view ABCC7 p.Met837* details
Enhanced basal halide efflux following the switch to the dequench buffer was detected in cells coexpressing
M837X
and ∆1-836 (p < 0.001), and regulated halide efflux was seen with expression of CFTR upon forskolin stimulation (p < 0.001).
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164
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:164:53
status:
NEW
view ABCC7 p.Met837* details
No enhanced halide movement was detected when either
M837X
or ∆1-836 was expressed alone.
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170
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:170:70
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:170:159
status:
NEW
view ABCC7 p.Met837* details
These results indicate that enhanced PKA-dependent phosphorylation of
M837X
is responsible for the high basal halide efflux produced following coexpression of
M837X
and ∆1-836.
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171
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:171:82
status:
NEW
view ABCC7 p.Met837* details
The Carboxy Region of the R-Domain Is Necessary for the Phosphorylation-Dependent
M837X
Mobility Shift and PKA-Dependent Halide Efflux.
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172
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:172:75
status:
NEW
view ABCC7 p.Met837* details
To examine R-domain residues that are necessary for the phosphorylation of
M837X
elicited by ∆1-836, we tested shortened CFTR truncations missing part or all of the R-domain for functional and biochemical interactions with ∆1-836.
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173
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:173:0
status:
NEW
view ABCC7 p.Gly723* details
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.
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174
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:174:84
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:174:6
status:
NEW
view ABCC7 p.Gly723* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:174:150
status:
NEW
view ABCC7 p.Gly723* details
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).
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175
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:175:42
status:
NEW
view ABCC7 p.Gly723* details
Furthermore, no molecular weight shift of
G723X
could be detected when cells were treated with forskolin prior to lysis (Figure 7B, lane 4).
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176
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:176:0
status:
NEW
view ABCC7 p.Gly723* details
G723X
and ∆1-836 expressed together produced constitutive (unregulated) activity (Figure 8A).
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179
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:179:0
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:179:189
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:179:136
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:179:145
status:
NEW
view ABCC7 p.Gly723* details
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).
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186
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:186:88
status:
NEW
view ABCC7 p.Met837* details
Enhanced phosphorylation of the R-domain was also observed when the first half of CFTR,
M837X
(CFTR truncated immediately after the R-domain), was treated with forskolin or coexpressed with ∆1-836.
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188
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:188:28
status:
NEW
view ABCC7 p.Gly723* details
A shorter amino truncation (
G723X
) also tightly bound to ∆1-836.
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189
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:189:15
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:189:159
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:189:161
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:189:22
status:
NEW
view ABCC7 p.Gly723* details
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 a
nd ∆1-836 is PKA-dependent.
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190
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:190:0
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:190:13
status:
NEW
view ABCC7 p.Met837* details
M837X
(2) or
M837X
and ∆1-836 (9) were expressed in COS7 cells.
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191
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:191:17
status:
NEW
view ABCC7 p.Met837* details
Cells expressing
M837X
and ∆1-836 were treated with Rp-8-CPT-cAMPS (b), a PKA antagonist.
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194
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:194:65
status:
NEW
view ABCC7 p.Met837* details
Rp-8-CPT-cAMPS dramatically inhibited the halide permeability of
M837X
and ∆1-836 (p < 0.05).
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195
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:195:97
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:195:112
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:195:148
status:
NEW
view ABCC7 p.Met837* details
Permeability coefficients (average changes in flourescence per 20 s from 80 to 120 s) were -2.2 (
M837X
), -12.3 (
M837X
and ∆1-836), and -3.9 (
M837X
and ∆1-836, with Rp-8CPT-cAMPS treatment).
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197
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:197:10
status:
NEW
view ABCC7 p.Gly723* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:197:94
status:
NEW
view ABCC7 p.Gly723* details
FIGURE 7:
G723X
binds ∆1-836, but this interaction does not elicit a mobility shift of
G723X
.
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199
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:199:21
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:199:87
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:199:11
status:
NEW
view ABCC7 p.Gly723* details
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).
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200
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:200:21
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:200:30
status:
NEW
view ABCC7 p.Gly723* details
(B) Cells expressing
M837X
or
G723X
were treated with forskolin prior to lysis (as described in the legend of Figure 4).
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201
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:201:0
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:201:88
status:
NEW
view ABCC7 p.Met837* details
M837X
from cells treated with forskolin appeared as an apparent higher-molecular weight
M837X
-CFTR protein band (*, lane 2).
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202
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:202:48
status:
NEW
view ABCC7 p.Gly723* details
However, forskolin treament of cells expressing
G723X
did not result in a higher-molecular weight protein (lane 4).
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207
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:207:0
status:
NEW
view ABCC7 p.Met837* details
M837X
coexpressed with ∆1-836 resulted in high-level CFTR activity (as detected by SPQ, Figure 5) in the absence of additional PKA stimulation, indicating that R-domain interactions with downstream domains can augment R-domain phosphorylation and directly regulate CFTR function.
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209
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:209:53
status:
NEW
view ABCC7 p.Met837* details
In contrast to ∆R-CFTR, the basal activity of
M837X
with ∆1-836 reported in this study was PKA-dependent, and could be inhibited by the PKA antagonist Rp-8-CPT-cAMPS.
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210
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:210:117
status:
NEW
view ABCC7 p.Met837* details
Therefore, while ∆R-CFTR functions independently of PKA, the high basal activity conferred by coexpression of
M837X
with ∆1-836 results from a different, phosphorylation-dependent mechanism.
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211
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:211:0
status:
NEW
view ABCC7 p.Gly723* details
G723X
also produced high basal halide efflux when it was coexpressed with ∆1-836 (Figure 8A).
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212
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:212:24
status:
NEW
view ABCC7 p.Met837* details
However, in contrast to
M837X
, the activity could not be inhibited by PKA antagonists (Figure 8B), a result similar to the activity reported for ∆R-CFTR.
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213
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:213:109
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:213:247
status:
NEW
view ABCC7 p.Met837* details
On the basis of the observations that (1) the level of the phosphorylation-dependent higher-molecular weight
M837X
was dramatically increased when it was bound to ∆1-836, (2) a high basal halide permeability was produced by coexpression of
M837X
with ∆1-836, and (3) this activity was suppressed by PKA inhibitors, we conclude that R-domain phosphorylation and CFTR activation were promoted by R-domain interactions with downstream elements of CFTR.
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214
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:214:102
status:
NEW
view ABCC7 p.Met837* details
The increased susceptibility of the R-domain to PKA phosphorylation in the two-subunit model of CFTR (
M837X
and ∆1-836) may reflect greater accessibility of the R-domain to endogenous PKA, or blockade of phosphatase action.
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228
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:228:87
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:228:10
status:
NEW
view ABCC7 p.Gly723* details
FIGURE 8:
G723X
coexpression with ∆1-836 produces enhanced halide efflux, while
K593X
does not.
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229
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:229:35
status:
NEW
view ABCC7 p.Lys593* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:229:4
status:
NEW
view ABCC7 p.Gly723* details
(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.
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230
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:230:112
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:230:0
status:
NEW
view ABCC7 p.Gly723* details
G723X
produced high basal halide permeability when coexpressed with ∆1-836 (p < 0.01) similar to that of
M837X
with ∆1-836 (Figure 5).
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231
ABCC7 p.Lys593*
X
ABCC7 p.Lys593* 10933805:231:0
status:
NEW
view ABCC7 p.Lys593* details
K593X
, missing the complete R-domain, failed to increase halide permeability when coexpressed with ∆1-836.
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234
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:234:4
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:234:35
status:
NEW
view ABCC7 p.Gly723* details
(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.
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235
ABCC7 p.Met837*
X
ABCC7 p.Met837* 10933805:235:62
status:
NEW
view ABCC7 p.Met837* details
ABCC7 p.Gly723*
X
ABCC7 p.Gly723* 10933805:235:117
status:
NEW
view ABCC7 p.Gly723* details
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).
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