The early events that occur rapidly after injury trigger signal cascades that are essential for proper wound closure of corneal epithelial cells. We hypothesize that injury releases ATP, which stimulates purinergic receptors and elicits the phosphorylation of epidermal growth factor receptor (EGFR) tyrosine residues and subsequent cell migration by a MMP and HB-EGF dependent pathway. We demonstrated that the inhibition of purinergic receptors with the antagonist, Reactive Blue 2, abrogated the phosphorylation of EGFR and ERK. Pre-incubation of cells with the EGFR kinase inhibitor, AG1478, and subsequent stimulation by injury or ATP resulted in a decrease in phosphorylation of EGFR and migration. Furthermore, downregulation of EGFR by siRNA, inhibited the EGF-induced intracellular Ca(2+) wave. However, the response to injury and ATP was retained indicating the presence of two signaling pathways. Inhibition with either CRM197 or TIMP-3 decreased injury and nucleotide-induced phosphorylation of both EGFR and ERK. Incubation in the presence of a functional blocking antibody to HB-EGF also resulted in a decrease in the phosphorylation of EGFR. In addition, cell migration was inhibited by CRM197 and rescued when cells were incubated with HB-EGF. We showed that injury-induced phosphorylation of specific tyrosine residues and found that a similar pattern of phosphorylation was induced by trinucleotides. These studies indicate that injury-induced purinergic receptor activation leads to phosphorylation of EGFR, ERK and migration.