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