A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.
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.
Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.Show MeSH
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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).
Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.