Blackhart B D, Cuenco G, Toda T, Scarborough R M, Wolf D L, Ramakrishnan V
COR Therapeutics, Inc., South San Francisco, California 94080.
Growth Factors. 1994;11(1):17-28. doi: 10.3109/08977199409015048.
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., Wheaton, 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 EC50 of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells. Binding of 125I-alpha-thrombin to clone #5-18 did not reach equilibrium at 37 degrees C. However, direct binding studies of 125I-alpha-, 125I-diisopropylphospho (DIP)-alpha-, and 125I-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 Kd's of 0.8 nM, 0.7 nM and 9.7 nM for 125I-alpha-, 125I-DIP-alpha- and 125I-beta-thrombin respectively. Association of 125I-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 125I-alpha- and 125I-DIP-alpha-thrombin, but did compete for 125I-beta-thrombin. In addition, the hirudin54-65 peptide competed at submicromolar concentrations for the binding of alpha- and DIP-alpha-thrombin, but not for beta-thrombin. This peptide binds specifically at the anion-binding exosite of alpha-thrombin and has been shown to have a lower affinity for beta-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.
凝血酶受体已被证明是G蛋白偶联受体家族的一个新成员（Vu，T.-K.H.，Hung，D.T.，Wheaton，V.I.和Coughlin，S.R.（1991年）《细胞》64卷，1057 - 1068页）。该受体似乎通过凝血酶介导的蛋白水解机制被激活，该机制会暴露出负责受体激活的“拴系配体”。为了研究凝血酶与该受体的初始相互作用，我们构建了表达高水平人凝血酶受体的细胞系，并研究了各种形式的凝血酶与细胞表面的结合。用凝血酶受体单克隆抗体分析转染细胞，鉴定出一个特定的细胞系（克隆#5 - 18），每个细胞显示有超过150,000个凝血酶受体。克隆#5 - 18似乎表达功能性受体，因为与未转染的CHO细胞相比，用凝血酶处理导致细胞质磷酸肌醇水平增加15 - 20倍，并且凝血酶介导的钙动员的EC50有类似的变化。125I-α-凝血酶与克隆#5 - 18的结合在37℃时未达到平衡。然而，125I-α-、125I-二异丙基磷酸（DIP）-α-和125I-β-凝血酶与克隆#5 - 18的直接结合研究表明，在4℃时每种配体的结合是可饱和且可逆的。结合数据分析显示，125I-α-、125I-DIP-α-和125I-β-凝血酶的Kd值分别为0.8 nM、0.7 nM和9.7 nM。125I-α-、DIP-α-和β-凝血酶的结合可以被未标记的α-和DIP-α-凝血酶竞争。未标记的β-凝血酶具有修饰的阴离子结合外位点，是125I-α-和125I-DIP-α-凝血酶的弱竞争者，但能竞争125I-β-凝血酶。此外，水蛭素54 - 65肽在亚微摩尔浓度下竞争α-和DIP-α-凝血酶的结合，但不竞争β-凝血酶的结合。该肽特异性结合于α-凝血酶的阴离子结合外位点，并且已显示对β-凝血酶的亲和力较低。这些结果直接证明了凝血酶与其受体之间的高亲和力相互作用，并表明一个重要组成部分是凝血酶受体与凝血酶阴离子结合外位点的高亲和力结合。
