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: To provide further evidence that ADAM17 is required for EGFR transactivation, we exploited our previously described in situ viral transduction approach (Khandelwal et al., 2008, 2010), in this case to express ADAM17-specific short hairpin RNAs (shRNAs) or scrambled shRNAs in the rat bladder uroepithelium. We used rat bladders in these studies because the volume capacity of the bladder, and therefore the number of virus particles needed, was relatively small (∼500 μl) compared with the volumes required to fill the rabbit bladder (∼60–100 ml). This technique targets the umbrella cell layer and achieves transduction efficiencies of 70–95% (Khandelwal et al., 2008, 2010). Indeed, we were able to achieve >90% knockdown of ADAM17 expression (Figure 4, A and B). On examining the expression and distribution of ADAM17 in cross sections of uroepithelium, we observed that knockdown of ADAM17 was largely confined to the umbrella cells, and expression of ADAM17 in the underlying cell layers was largely undisturbed (Figure 4C).
Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.