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V. Trinkaus–Randall, L. Yang, I. Boucher; Injury and Trinucleotides Transactivate Specific Tyrosine Residues of the EGF Receptor Leading to Downstream Signaling . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5020.
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Previously we demonstrated that trinucleotides released from the wound stimulate purinergic receptors and elicit a complex signaling cascade that ultimately mediates wound closure.
Our goal is to evaluate the role and activation of the epidermal growth factor receptor (EGFR) tyrosine residues that occur in response to injury induced purinergic receptor activation.
Primary corneal epithelial cells and HCE–Ts were used for evaluation. Calcium signaling was monitored using live cell confocal imaging with a perfusion system. Phosphorylation and localization of specific EGFR tyrosine residues was assessed using immunohistochemistry, confocal microscopy and western blot analysis. The EGFR was downregulated using a kinase inhibitor AG1478 or siRNA.
We have demonstrated that downregulation of EGFR via siRNA or kinase inhibitors reduces injury induced ERK phosphorylation. Injury induced a differential phosphorylation of the EGFR on tyrosine residues,1068, 1086 and 1173, compared to that of EGF. Phosphorylation of tyrosine residues 845 and 1148 was not detected. Phosphorylation of pY1086 and pY1068 was most intense along the wound edge. Cells stimulated with trinucleotides showed a similar differential phosphorylation as cells that were wounded. However, cells stimulated with dinucleotides showed an altered response. Uridine diphosphate (UDP) (an agonist for the purinergic receptor, P2Y6) did not stimulate phosphorylation of any of the tyrosine residues evaluated. The use of CRM 197 and/or TIMP–3 to inhibit heparin binding–EGF (HB–EGF) specifically decreased the injury induced phosphorylation of EGFR and ERK. Interestingly, pY1068 and 1086 were the only sites that were significantly reduced. Stimulation with EGF was not altered in the presence of TIMP–3 or CRM 197.
These results indicate that injury activates trinucleotide receptors resulting in the release of HB–EGF and phosphorylation of specific tyrosine residues on EGFR that ultimately mediates repair.
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