BINDING OF BIOTINYLATED THROMBIN RECEPTOR PEPTIDE TO CLONED HUMAN THROMBIN RECEPTOR OVEREXPRESSED IN BABY HAMSTER-KIDNEY CELLS

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblas t growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Ana lysis of the binding to live BHK cells yielded an equilibrium dissocia tion constant (Kd) of 3.0+/-0.3 mu mol/l and a maximal binding capacit y (Bmax) of 31.0+/-0.5 nmol/mg of protein. In competitive binding expe riments, the thrombin receptor agonist peptide (SFLLRN), which is a st rong inducer of human platelet aggregation, was the most potent compet itor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPND KYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system usin g the biotinylated thrombin receptor peptide as a labeled ligand and t he cloned thrombin receptor overexpressed in BHK cells may be useful f or exploring specific antagonists of the receptor.