AbstractThe thrombin receptor has been shown to be a novel member of the family of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991)Cell64, 1057–1068). This receptor appears to be activated through a thrombin-mediated proteolytic mechanism which exposes a“tethered ligand”responsible for receptor activation. In order to investigate the initial interactions of thrombin with this receptor, we have constructed cell lines which express high levels of the human thrombin receptor and studied the binding of various forms of thrombin to the cell surface. Analysis of transfected cells with thrombin receptor monoclonal antibodies identified a particular cell line (clone #5–18) which displayed>150,000 thrombin receptors per cell. Clone #5–18 appeared to express functional receptors since treatment with thrombin resulted in both a 15–20 fold increase of cytoplasmic phosphoinositide levels and a comparable shift in the EC50of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells.Binding of125I-α-thrombin to clone #5–18 did not reach equilibrium at 37°C. However, direct binding studies of125I-α-,125I-diisopropylphospho (DIP)-α-, and125I-β-thrombin to clone #5–18 demonstrated that binding at 4°C was saturable and reversible for each ligand. Analysis of the binding data revealed Kd's of 0.8 nM, 0.7 nM and 9.7 nM for125I-α-,125I-DIP-α- and125I-β-thrombin respectively. Association of125I-α-, DIP-α, andβ-thrombin could be competed by unlabelledα- and DIP-α-thrombin. Unlabelledβ-thrombin, which has a modified anion-binding exosite, was a poor competitor for125I-α-and125I-DIP-α-thrombin, but did compete for125I-β-thrombin. In addition, the hirudin54–65peptide competed at submicromolar concentrations for the binding ofα- and DIP-α-thrombin, but not forβ-thrombin. This peptide binds specifically at the anion-binding exosite ofα-thrombin and has been shown to have a lower affinity forβ-thrombin. These results demonstrate directly a high affinity interaction between thrombin and its receptor, and suggest that an important component is the high affinity association of the thrombin receptor with the anion-binding exosite of thrombin.