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A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

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Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are marked with arrows. (B) HEK-293FT cells expressing the A1AR in combination with ADAM17-HAr or ADAM17S811A-HAr were labeled with 32P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect 32P-labeled ADAM17 (top). Total A1AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantified, and values significantly different from ADAM17-HAr + CCPA are indicated with an asterisk. (D) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data significantly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr. Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (*p < 0.05). (F) Rat tissues were transduced in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk.
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Figure 6: Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are marked with arrows. (B) HEK-293FT cells expressing the A1AR in combination with ADAM17-HAr or ADAM17S811A-HAr were labeled with 32P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect 32P-labeled ADAM17 (top). Total A1AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantified, and values significantly different from ADAM17-HAr + CCPA are indicated with an asterisk. (D) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data significantly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr. Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (*p < 0.05). (F) Rat tissues were transduced in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk.

Mentions: Next we used ADAM17-specific shRNAs to test whether ADAM17 was required for CCPA-stimulated EGFR transactivation and exocytosis in rat epithelium. Treatment with ADAM17-shRNA, but not scrambled shRNA, decreased EGFR phosphorylation (Figure 4, D and E). We show later in Figure 6, F and G, that the effects of the ADAM17-shRNA are specific, and ADAM17 function is restored when the shRNA is coexpressed with shRNA-resistant variant of wild-type ADAM17. We also attempted to measure changes in CT in the rat bladders treated with apical CCPA; however, we did not observe a response. Because CT is dependent on the rates of membrane addition and removal, we reasoned that one possible explanation was that CCPA stimulated both exocytosis and endocytosis in rat epithelium, thus obviating any change in apparent CT. Indeed, we observed that wheat germ agglutinin–fluorescein isothiocyanate (WGA-FITC), added to the apical hemichamber of mounted rat bladders, was endocytosed in CCPA-treated tissue but much less so in control, untreated tissue (Figure 4F). To circumvent the effects of endocytosis, we instead measured release of exogenously expressed human growth hormone (hGH), which we and others previously showed is packaged into DFVs and released from the luminal surface of the bladder into the urinary space (Kerr et al., 1998; Khandelwal et al., 2008). Because of rapid dilution, there is little secreted hGH that is endocytosed. Compared to control bladders, CCPA stimulated a large increase in the mucosal release of hGH from the tissue (Figure 4, G and H). Moreover, expression of ADAM-17-specific shRNA, but not scrambled shRNA, caused a large inhibition (>90%) in mucosal hGH release (Figure 4, G and H).


A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are marked with arrows. (B) HEK-293FT cells expressing the A1AR in combination with ADAM17-HAr or ADAM17S811A-HAr were labeled with 32P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect 32P-labeled ADAM17 (top). Total A1AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantified, and values significantly different from ADAM17-HAr + CCPA are indicated with an asterisk. (D) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data significantly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr. Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (*p < 0.05). (F) Rat tissues were transduced in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk.
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Figure 6: Effect of Ser-811 phosphorylation on the function of ADAM17. (A) Top, alignment of the C-termini of ADAM17 from different species. Numbers indicate the amino acids involved. The canonical PKC phosphorylation site is shaded, and the position of the conserved Ser residue (Ser-811 in rat) is indicated in red. Bottom, domain structure of ADAM17. CR, cysteine-rich domain; C-tail, cytoplasmic domain; Dis, disintegrin domain; EL, EGF-like; MP, metalloproteinase domain; Pro, propeptide; TM, transmembrane domain. The consensus PKC phosphorylation motif is shown in an expanded view. Ser-811 is shaded, and the critical, flanking basic residues at the −3 and + 2 positions are marked with arrows. (B) HEK-293FT cells expressing the A1AR in combination with ADAM17-HAr or ADAM17S811A-HAr were labeled with 32P-orthophosphate and then left untreated or treated with CCPA (500 nM). HA-tagged ADAM17 constructs were immunoprecipitated and Western blots probed with an anti–HA-HRP- conjugated secondary antibody to detect total ADAM17 (middle) and subsequently exposed to a PhosphorImager screen to detect 32P-labeled ADAM17 (top). Total A1AR was detected by Western blot (bottom). (C) Data (mean ± SEM; n = 3) were quantified, and values significantly different from ADAM17-HAr + CCPA are indicated with an asterisk. (D) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. Fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells. Data are mean ± SEM, n = 5. Data significantly different from HB-EGF-AP alone are indicated with an asterisk. (E) HEK-293FT cells were cotransfected with the A1AR, HB-EGF-AP, and ADAM17-HAr. Cells were pretreated with calphostin C (500 nM) for 60 min, and the fold stimulation of HB-EGF-AP release in CCPA-treated cells vs. control, untreated cells is reported. Data are mean ± SEM, n = 7. Values significantly different from the control, as assessed by ANOVA, are indicated (*p < 0.05). (F) Rat tissues were transduced in situ with hGH and ADAM17 shRNA alone or in combination with ADAM17-HAr, ADAM17S811A-HAr, or ADAM17S811D-HAr. The excised bladder tissue was mounted in an Ussing stretch chamber, and CCPA (500 nM) was added to the mucosal hemichamber. After 60 min, the mucosal fluid was collected and concentrated, the tissues were lysed, and hGH detected using Western blot. (G) Quantification of hGH secretion. Data are mean ± SEM (n = 5). Values that are statistically different from ADAM17 shRNA alone (p < 0.05) are indicated with an asterisk.
Mentions: Next we used ADAM17-specific shRNAs to test whether ADAM17 was required for CCPA-stimulated EGFR transactivation and exocytosis in rat epithelium. Treatment with ADAM17-shRNA, but not scrambled shRNA, decreased EGFR phosphorylation (Figure 4, D and E). We show later in Figure 6, F and G, that the effects of the ADAM17-shRNA are specific, and ADAM17 function is restored when the shRNA is coexpressed with shRNA-resistant variant of wild-type ADAM17. We also attempted to measure changes in CT in the rat bladders treated with apical CCPA; however, we did not observe a response. Because CT is dependent on the rates of membrane addition and removal, we reasoned that one possible explanation was that CCPA stimulated both exocytosis and endocytosis in rat epithelium, thus obviating any change in apparent CT. Indeed, we observed that wheat germ agglutinin–fluorescein isothiocyanate (WGA-FITC), added to the apical hemichamber of mounted rat bladders, was endocytosed in CCPA-treated tissue but much less so in control, untreated tissue (Figure 4F). To circumvent the effects of endocytosis, we instead measured release of exogenously expressed human growth hormone (hGH), which we and others previously showed is packaged into DFVs and released from the luminal surface of the bladder into the urinary space (Kerr et al., 1998; Khandelwal et al., 2008). Because of rapid dilution, there is little secreted hGH that is endocytosed. Compared to control bladders, CCPA stimulated a large increase in the mucosal release of hGH from the tissue (Figure 4, G and H). Moreover, expression of ADAM-17-specific shRNA, but not scrambled shRNA, caused a large inhibition (>90%) in mucosal hGH release (Figure 4, G and H).

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus