C-terminus of ADAM 17 is required for substrate cleavage

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To evaluate the importance of ADAM17 phosphorylation in the TMPS cascade leading to EGFR transactivation, the ability of unstimulated AGS gastric epithelial cells, AGS cells over expressing  wild type ADAM17 or ΔC-terminus ADAM17 to cleave ADAM17 substrates was determined using an IRDye Near Infrared Fluorescence Resonance Energy Transfer assay (NIR-FRET). The cleavage of HB-EGF and TNF-α quenched probes by the AGS cell lines was examined. AGS ΔC-terminus ADAM17 induced a small but significant (p < 0.05) increase in HB-EGF cleavage compared to non-transfected AGS cells. In contrast, AGS cells over expressing ADAM17 significantly increased (p < 0.05) HB-EGF cleavage relative to both non-transfected AGS cells and AGS ΔC-terminus ADAM17 cells (Figure 3A). Analysis of cleavage of the ADAM17 substrate TNF-α using a TNF-α quenched probe showed that the AGS ΔC-terminus ADAM17 cell line was able to significantly (p < 0.05) increase TNF-α cleavage relative to that induced by non-transfected gastric epithelial AGS cells. However cleavage induced by the AGS ΔC-terminus ADAM17 cell line was significantly lower (p < 0.05)  than that observed with AGS cells over expressing wild type ADAM17 (Figure 3B).

 To investigate further the importance of the ADAM17 cytoplasmic tail and its phosphorylation sites in autocrine cell signalling, HB-EGF and EGFR transcripts in unstimulated AGS cells, AGS cells overexpressing ADAM17 and AGS cells with ΔC-terminus ADAM17 were examined. Transfected cells were examined after culture with, and with out, tetracyline. Both ADAM17 over expressing wild type and ΔC-terminus ADAM17 AGS cells had significantly (p < 0.01) increased abundance of HB-EGF and EGFR transcripts in the absence of tetracycline compared to AGS epithelial cells (Figure 4A and B).  However both HB-EGF and EGFR transcript abundance was significantly (p < 0.05) higher in AGS cells over expressing ADAM17 than AGS cells with ΔC-terminus ADAM17 in cells cultured in the absence of tetracycline (Figure 4A and B).  This demonstrates that ADAM17 over expression in gastric epithelial cells in the absence of H. pylori stimulation will upregulate key components of the TMPS pathway promoting autocrine EGFR signalling.