ABCMdb

ABCC7 p.Met837*

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[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.

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No. Sentence Comment
4 A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with ∆1-836. Login to comment
5 Moreover, coexpression of M837X and ∆1-836 led to enhanced halide permeability in living cells. Login to 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. Login to comment
52 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). Login to comment
54 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). Login to comment
111 Interactions between ∆1-836 and M837X Confer Phosphorylation of the R-Domain and ActiVation of Halide Efflux. Login to comment
112 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). Login to comment
115 Immunoprecipitates from COS7 cells expressing M837X, ∆1-836, or both were studied by Western blotting, as shown in Figure 3. Login to comment
116 The M837X protein was pulled down in association with ∆1-836 (Figure 3A). Login to comment
117 The complementary experiment showed that ∆1-836 could also be co-immunoprecipitated in association with M837X (Figure 3B, lane 6). Login to comment
118 Neither M837X nor ∆1-836 bound to a control ( -gal) protein (Figure 3C). Login to comment
119 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 *). Login to comment
120 Direct immunoprecipitation of M837X indicated that the total level of shifted M837X protein was negligible when expressed alone (Figure 3A, lane 1). Login to comment
121 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). Login to comment
122 To test whether phosphorylation was responsible for the mobility shift in M837X, cells expressing M837X alone were treated with forskolin prior to lysis. Login to comment
123 Forskolin treatment reproduced the slower molecular weight migration in M837X (Figure 4A, lane 1). Login to comment
124 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). Login to comment
125 These results indicate that the expression of ∆1-836 with M837X confers a phosphorylation-dependent reduction in the mobility of M837X. Login to comment
126 Forskolin treatment did not cause phosphorylation of all the expressed R-domain or M837X. Login to comment
129 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. Login to comment
132 Since we observed that binding between M837X and ∆1-836 augments CFTR phosphorylation, we tested the functional consequences of this binding interaction. Login to comment
133 M837X, ∆1-836, or both were expressed in COS7 cells, and anion efflux was measured using the halide sensitive dye SPQ. Login to comment
134 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. Login to comment
141 background was detected in cells expressing either M837X or ∆1-836 alone (Figure 5, 0 and O). Login to comment
142 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). Login to comment
146 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. Login to comment
148 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. Login to comment
149 M837X, ∆1-836, or both were immunoprecipitated using an antibody specific to M837X, or to the C-terminal half. Login to comment
150 M837X co-immunoprecipitated with ∆1-836 (panel A, lane 4). Login to comment
151 The ∆1-836 was also detected following co-immunoprecipitation with M837X (panel B, lane 6). Login to comment
153 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. Login to comment
154 FIGURE 4: Forskolin treatment or coexpression with ∆1-836 produces an apparent higher-molecular weight, alkaline phosphatase sensitive form of M837X. Login to comment
155 Lysates from cells expressing M837X, alone or together with ∆1-836 ("both"), were immunoprecipitated as described in the legend of Figure 3. Login to comment
157 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). Login to comment
158 The higher-molecular weight M837X protein band was eliminated by treatment with alkaline phosphatase (lanes 2 and 4). Login to comment
159 FIGURE 5: M837X and ∆1-836 coexpression produces elevated basal halide efflux. Login to comment
160 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. Login to comment
163 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). Login to comment
164 No enhanced halide movement was detected when either M837X or ∆1-836 was expressed alone. Login to comment
170 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. Login to comment
171 The Carboxy Region of the R-Domain Is Necessary for the Phosphorylation-Dependent M837X Mobility Shift and PKA-Dependent Halide Efflux. Login to comment
172 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. Login to comment
174 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). Login to comment
179 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). Login to comment
186 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. Login to comment
189 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. Login to comment
190 M837X (2) or M837X and ∆1-836 (9) were expressed in COS7 cells. Login to comment
191 Cells expressing M837X and ∆1-836 were treated with Rp-8-CPT-cAMPS (b), a PKA antagonist. Login to comment
194 Rp-8-CPT-cAMPS dramatically inhibited the halide permeability of M837X and ∆1-836 (p < 0.05). Login to comment
195 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). Login to comment
199 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). Login to comment
200 (B) Cells expressing M837X or G723X were treated with forskolin prior to lysis (as described in the legend of Figure 4). Login to comment
201 M837X from cells treated with forskolin appeared as an apparent higher-molecular weight M837X-CFTR protein band (*, lane 2). Login to comment
207 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. Login to comment
209 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. Login to comment
210 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. Login to comment
212 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. Login to comment
213 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. Login to comment
214 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. Login to comment
230 G723X produced high basal halide permeability when coexpressed with ∆1-836 (p < 0.01) similar to that of M837X with ∆1-836 (Figure 5). Login to comment
234 (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. Login to comment
235 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). Login to comment