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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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Role for Gi, Gβγ, PLC, and PKC in A1AR-stimulated exocytosis. (A) Rabbit tissue was left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for 60 min, or 10 μM U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, and CT was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk.
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Figure 5: Role for Gi, Gβγ, PLC, and PKC in A1AR-stimulated exocytosis. (A) Rabbit tissue was left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for 60 min, or 10 μM U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, and CT was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk.

Mentions: We next addressed how ADAM17 activity is coupled to A1AR activation. Previous studies showed that A1AR signals through Giα to inhibit the activity of adenylyl cyclase, whereas the βγ subunits of Gi increase the activity of phospholipase C-β (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol trisphosphate (IP3) and diacylgycerol (Freund et al., 1994; Bucheimer and Linden, 2004; Chang et al., 2008). The latter stimulates the activity of PKC, a well-known regulatory kinase that was previously implicated in ADAM17 activation (Dang et al., 2011; Kveiborg et al., 2011; Lemjabbar-Alaoui et al., 2011). We found that pertussis toxin–mediated inhibition of Gi or inhibition of Gβγ subunit activity by the inhibitor M119K (Kirui et al., 2010) significantly impaired CCPA-mediated apical exocytosis in rabbit bladder umbrella cells (Figure 5A). Furthermore, the PLC-selective antagonist U73122 caused marked inhibition of CCPA-induced changes in CT (Figure 5A). Thus ADAM17 activation downstream of A1AR likely occurs by way of a classical Gi-stimulated signaling cascade involving Gβγ and PLCβ.


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)

Role for Gi, Gβγ, PLC, and PKC in A1AR-stimulated exocytosis. (A) Rabbit tissue was left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for 60 min, or 10 μM U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, and CT was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk.
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Figure 5: Role for Gi, Gβγ, PLC, and PKC in A1AR-stimulated exocytosis. (A) Rabbit tissue was left untreated or pretreated with 100 ng/ml pertussis toxin (PTX) for 90 min, 10 μM M119K for 60 min, or 10 μM U73122 for 60 min. CCPA (500 nM) was added to the mucosal hemichamber, and CT was recorded. (B) Rabbit tissue was left untreated or pretreated with calphostin C (500 nM) for 90 min. Subsequently, tissue was treated with CCPA (500 nM) or PMA (10 nM). (C) Rabbit tissue was pretreated with 15 μM Tapi-2 for 90 min or 1 μM AG1478 for 30 min and then treated with 10 nM PMA. The data for PMA treatment alone were reproduced from B. (A–C) Data for CCPA treatment alone were reproduced from Figure 1B. (D, E) Rabbit tissue was pretreated with calphostin C (D) or Tapi-2 (E) for 90 min, and then at t = 0, CCPA was added to the mucosal hemichamber. After 120 min, HG-EGF (1 nM) was added to the mucosal hemichamber (indicated with an arrow), and the tissue was incubated for additional 180 min. In D, data for calphostin C + CCPA are reproduced from B. (A–E) Data are presented as mean ± SEM (in A–C, n ≥ 3; in D and E, n ≥ 6). In A–C, values that are significantly different from CCPA alone (p < 0.05) are marked with an asterisk. In D and E, values that are significantly different from calphostin C + CCPA or Tapi-2 + CCPA (p < 0.05) are marked with an asterisk.
Mentions: We next addressed how ADAM17 activity is coupled to A1AR activation. Previous studies showed that A1AR signals through Giα to inhibit the activity of adenylyl cyclase, whereas the βγ subunits of Gi increase the activity of phospholipase C-β (PLCβ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol trisphosphate (IP3) and diacylgycerol (Freund et al., 1994; Bucheimer and Linden, 2004; Chang et al., 2008). The latter stimulates the activity of PKC, a well-known regulatory kinase that was previously implicated in ADAM17 activation (Dang et al., 2011; Kveiborg et al., 2011; Lemjabbar-Alaoui et al., 2011). We found that pertussis toxin–mediated inhibition of Gi or inhibition of Gβγ subunit activity by the inhibitor M119K (Kirui et al., 2010) significantly impaired CCPA-mediated apical exocytosis in rabbit bladder umbrella cells (Figure 5A). Furthermore, the PLC-selective antagonist U73122 caused marked inhibition of CCPA-induced changes in CT (Figure 5A). Thus ADAM17 activation downstream of A1AR likely occurs by way of a classical Gi-stimulated signaling cascade involving Gβγ and PLCβ.

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