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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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The A1AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 μM U0126 for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and CT was recorded. (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) was added to the mucosal hemichamber and CT was recorded. (A, B) CCPA control data are reproduced from Figure 1B. Mean changes in CT ± SEM (n ≥ 3). Statistically significant differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for 25 min, Tapi-2 (15 μM) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y1173 antibody or rabbit anti-EGFR antibody. (D) Quantification of Y1173 phosphorylation. Data (mean ± SEM, n ≥ 3) are reported as fold increase above untreated tissue samples at t = 0. Statistically significant values (p < 0.05) above t = 0 are marked by an asterisk.
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Figure 2: The A1AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 μM U0126 for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and CT was recorded. (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) was added to the mucosal hemichamber and CT was recorded. (A, B) CCPA control data are reproduced from Figure 1B. Mean changes in CT ± SEM (n ≥ 3). Statistically significant differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for 25 min, Tapi-2 (15 μM) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y1173 antibody or rabbit anti-EGFR antibody. (D) Quantification of Y1173 phosphorylation. Data (mean ± SEM, n ≥ 3) are reported as fold increase above untreated tissue samples at t = 0. Statistically significant values (p < 0.05) above t = 0 are marked by an asterisk.

Mentions: We also assessed whether CCPA triggered apical exocytosis by transactivating the EGFR. We first tested the effect of treating the mucosal surface of the tissue with CRM197, a mutant version of diphtheria toxin that binds selectively and with high affinity to membrane-bound HB-EGF and prevents its cleavage (Uchida et al., 1973). Indeed, we observed that CRM197 almost completely blocked CCPA-mediated apical exocytosis (Figure 2A). Next we pretreated tissue with AG1478, a small-molecule inhibitor of EGFR, which also significantly blocked CCPA-mediated apical exocytosis (Figure 2B). As further evidence that the EGFR was transactivated, we generated lysates from CCPA-treated epithelium and then probed Western blots with an antibody that detects phosphorylation of the EGFR at Tyr-1173. This residue promotes assembly of a MAPK signaling cascade downstream of other GPCRs (Balestreire and Apodaca, 2007). Phosphorylation of Tyr-1173 was maximally stimulated 10 min after continuous treatment with CCPA, decreased at 30 min posttreatment, and returned to control levels by 60 min (Figure 2, C and D). The Tyr-1173 phosphorylation was prevented when the tissue was pretreated with the EGFR inhibitor AG1478 before CCPA treatment (Figure 2, C and D), confirming that phosphorylation of Tyr-1173 likely results from autophosphorylation. Finally, we observed that U0126, an inhibitor of MEK activity, also caused a significant decrease in CCPA-stimulated changes in CT (Figure 2A).


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)

The A1AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 μM U0126 for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and CT was recorded. (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) was added to the mucosal hemichamber and CT was recorded. (A, B) CCPA control data are reproduced from Figure 1B. Mean changes in CT ± SEM (n ≥ 3). Statistically significant differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for 25 min, Tapi-2 (15 μM) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y1173 antibody or rabbit anti-EGFR antibody. (D) Quantification of Y1173 phosphorylation. Data (mean ± SEM, n ≥ 3) are reported as fold increase above untreated tissue samples at t = 0. Statistically significant values (p < 0.05) above t = 0 are marked by an asterisk.
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Related In: Results  -  Collection

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Figure 2: The A1AR transactivates the EGF receptor. (A) The mucosal surface of rabbit uroepithelium was pretreated with 25 ng/ml CRM197 for 25 min or with 10 μM U0126 for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber, and CT was recorded. (B) Rabbit uroepithelium was pretreated with 1 μM AG1478 for 30 min, and then CCPA (500 nM) was added to the mucosal hemichamber and CT was recorded. (A, B) CCPA control data are reproduced from Figure 1B. Mean changes in CT ± SEM (n ≥ 3). Statistically significant differences (p < 0.05), relative to CCPA treatment alone, are marked with an asterisk. (C, D) Rabbit uroepithelium was either left untreated (left) or treated with AG1478 (1 μM) for 25 min, Tapi-2 (15 μM) for 90 min, or calphostin C (500 nM) for 60 min (right). CCPA (500 nM) was then added to the mucosal hemichamber. Left, cells were lysed at the indicated time points. Right, cells were lysed at the 10-min time point. Equal amounts of proteins were resolved by SDS–PAGE and immunoblots probed with a rabbit anti–EGFR-phospho-Y1173 antibody or rabbit anti-EGFR antibody. (D) Quantification of Y1173 phosphorylation. Data (mean ± SEM, n ≥ 3) are reported as fold increase above untreated tissue samples at t = 0. Statistically significant values (p < 0.05) above t = 0 are marked by an asterisk.
Mentions: We also assessed whether CCPA triggered apical exocytosis by transactivating the EGFR. We first tested the effect of treating the mucosal surface of the tissue with CRM197, a mutant version of diphtheria toxin that binds selectively and with high affinity to membrane-bound HB-EGF and prevents its cleavage (Uchida et al., 1973). Indeed, we observed that CRM197 almost completely blocked CCPA-mediated apical exocytosis (Figure 2A). Next we pretreated tissue with AG1478, a small-molecule inhibitor of EGFR, which also significantly blocked CCPA-mediated apical exocytosis (Figure 2B). As further evidence that the EGFR was transactivated, we generated lysates from CCPA-treated epithelium and then probed Western blots with an antibody that detects phosphorylation of the EGFR at Tyr-1173. This residue promotes assembly of a MAPK signaling cascade downstream of other GPCRs (Balestreire and Apodaca, 2007). Phosphorylation of Tyr-1173 was maximally stimulated 10 min after continuous treatment with CCPA, decreased at 30 min posttreatment, and returned to control levels by 60 min (Figure 2, C and D). The Tyr-1173 phosphorylation was prevented when the tissue was pretreated with the EGFR inhibitor AG1478 before CCPA treatment (Figure 2, C and D), confirming that phosphorylation of Tyr-1173 likely results from autophosphorylation. Finally, we observed that U0126, an inhibitor of MEK activity, also caused a significant decrease in CCPA-stimulated changes in CT (Figure 2A).

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