THE ANION-BINDING EXOSITE IS CRITICAL FOR THE HIGH-AFFINITY BINDING OF THROMBIN TO THE HUMAN THROMBIN RECEPTOR

The 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., Wheaten, V.I., and Coughlin, S.R. (1991) Cell 64, 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 EC(50) of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells. Binding of I-125-alpha-thrombin to clone #5-18 did not reach equilibrium at 37 degrees C. However, direct binding studies of I-125-alpha-, I-125-diisopropylphospho (DIP)-alpha-, and I-125-beta-thrombin to clone #5-18 demonstrated that binding at 4 degrees C was saturable and reversible for each ligand. Analysis of the binding data revealed K-d's of 0.8 nM, 0.7 nM and 9.7 nM for I-125-DIP-alpha- and I-125-beta-thrombin respectively. Association of I-125-alpha-, DIP-alpha, and beta-thrombin could be competed by unlabelled alpha- and DIP-alpha-thrombin. Unlabelled beta-thrombin, which has a modified anion-binding exosite, was a poor competitor for I-125-DIP-alpha-thrombin, but did compete for I-125-beta-thrombin.