Thrombin is now recognized as an important factor in many cancers. Here, we examined the expression and role of the recently discovered thrombin receptor PAR4, in human colon cancer cells. PAR4 mRNA was found in 10 out of 14 (71%) human colon cancer cell lines tested but not in epithelial cells isolated from normal human colon. This finding is in line with immunostaining results of PAR4 in human colon tumors and its absence in normal human colonic mucosa. Investigation of the functional significance of the aberrant expression of PAR4 in colon cancer cells revealed (i) a prompt increase in intracellular calcium concentration on challenge with PAR4-specific agonist AP4 (100 microM) and (ii) marked mitogenic response (2.5-fold increase in cell number) in a dose-dependent manner on treatment with AP4 (0.1-300 microM). Analysis of the signaling pathways downstream of PAR4 activation in HT29 cells showed (i) a sustained phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and (ii) the involvement of epidermal growth factor receptor B-2 (ErbB-2) but not of epidermal growth factor receptor in PAR4-induced mitogen-activated protein kinase activation. Tyrphostin AG1478, the ErbB inhibitor, reversed the action of AP4 on ERK1/2 and ErbB-2 phosphorylation and HT29 cell growth. Finally, the Src inhibitor PP2 abrogated ErbB-2 and ERK phosphorylation and HT29 cell proliferation, suggesting the essential role of Src activity in PAR4-induced phosphorylation of ErbB-2. These data highlight the role of PAR4 as a new important player in the control of colon tumors and underline the critical role of ErbB-2 transactivation.