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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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ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder uroepithelium were reacted with antibodies specific for ADAM17 (A), a mixture of antibody and inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the apicolateral junction is indicated by arrowheads; scale bar, 10 μm. (D) Rabbit uroepithelium was pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and CT was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), and values significantly different (p < 0.05) from CCPA alone are marked with an asterisk.
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Figure 3: ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder uroepithelium were reacted with antibodies specific for ADAM17 (A), a mixture of antibody and inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the apicolateral junction is indicated by arrowheads; scale bar, 10 μm. (D) Rabbit uroepithelium was pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and CT was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), and values significantly different (p < 0.05) from CCPA alone are marked with an asterisk.

Mentions: In vivo studies implicate ADAM17 as the physiologically relevant sheddase in HB-EGF–mediated transactivation (Jackson et al., 2003; Sahin et al., 2004). Consistent with these observations, we observed that ADAM17 was localized to small vesicular elements under the apical surface of the rat umbrella cells (Figure 3A), the likely site of HB-EGF cleavage and EGFR transactivation during the late-phase response (Balestreire and Apodaca, 2007). Rat tissues were used in these experiments because our antibody was produced in rabbits. ADAM17 was also detected in the intermediate and basal cell layers of the uroepithelium, as well as in cells in the underlying lamina propria (Figure 3, A and C). The signal for ADAM17 was diminished when the anti-ADAM17 antibody was preincubated with immunizing peptide, confirming the specificity of the antibody (Figure 3B). ADAM17 showed a high degree of colocalization with uroplakin 3a (Manders coefficient of colocalization, 0.92); this is associated with DFVs and the apical surface of umbrella cells (Figure 3C; Apodaca, 2004). Consistent with a role for ADAM17 in DFV trafficking, we observed that the broad-spectrum metalloproteinase inhibitor GM6001 and the ADAM17-selective inhibitor Tapi-2 (Balestreire and Apodaca, 2007; Kveiborg et al., 2011) both significantly inhibited the CCPA-mediated increases in CT (Figure 3D). Moreover, Tapi-2 blocked CCPA-mediated phosphorylation of EGFR Tyr-1173 (Figure 2, C and D).


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)

ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder uroepithelium were reacted with antibodies specific for ADAM17 (A), a mixture of antibody and inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the apicolateral junction is indicated by arrowheads; scale bar, 10 μm. (D) Rabbit uroepithelium was pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and CT was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), and values significantly different (p < 0.05) from CCPA alone are marked with an asterisk.
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Figure 3: ADAM17 expression in the uroepithelium. (A–C) Cryosections of rat bladder uroepithelium were reacted with antibodies specific for ADAM17 (A), a mixture of antibody and inhibitory peptide (B), or antibodies specific for ADAM17 and uroplakin 3a (C). After incubation with fluorophore-labeled secondary antibodies, the samples were analyzed using confocal microscopy. Where indicated, actin was labeled with phalloidin and nuclei were labeled with TOPRO-3. The umbrella cells are marked with the asterisk in the merged images and the apicolateral junction is indicated by arrowheads; scale bar, 10 μm. (D) Rabbit uroepithelium was pretreated with Tapi-2 (15 μM) or GM6001 (15 μM) for 90 min, CCPA (500 nM) was added, and CT was recorded. Control CCPA data are reproduced from Figure 1B. Data are mean ±SEM (n ≥ 3), and values significantly different (p < 0.05) from CCPA alone are marked with an asterisk.
Mentions: In vivo studies implicate ADAM17 as the physiologically relevant sheddase in HB-EGF–mediated transactivation (Jackson et al., 2003; Sahin et al., 2004). Consistent with these observations, we observed that ADAM17 was localized to small vesicular elements under the apical surface of the rat umbrella cells (Figure 3A), the likely site of HB-EGF cleavage and EGFR transactivation during the late-phase response (Balestreire and Apodaca, 2007). Rat tissues were used in these experiments because our antibody was produced in rabbits. ADAM17 was also detected in the intermediate and basal cell layers of the uroepithelium, as well as in cells in the underlying lamina propria (Figure 3, A and C). The signal for ADAM17 was diminished when the anti-ADAM17 antibody was preincubated with immunizing peptide, confirming the specificity of the antibody (Figure 3B). ADAM17 showed a high degree of colocalization with uroplakin 3a (Manders coefficient of colocalization, 0.92); this is associated with DFVs and the apical surface of umbrella cells (Figure 3C; Apodaca, 2004). Consistent with a role for ADAM17 in DFV trafficking, we observed that the broad-spectrum metalloproteinase inhibitor GM6001 and the ADAM17-selective inhibitor Tapi-2 (Balestreire and Apodaca, 2007; Kveiborg et al., 2011) both significantly inhibited the CCPA-mediated increases in CT (Figure 3D). Moreover, Tapi-2 blocked CCPA-mediated phosphorylation of EGFR Tyr-1173 (Figure 2, C and D).

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