Thrombin Receptor Aromatic Residues for Edge-to-Face CH/π Interaction with Ligand Phe-2-phenyl Group

Serine protease thrombin plays an important role in blood coagulation and possesses a specific receptor in the platelets. When thrombin cleaves the peptide bond between Arg-41 and Ser-42 in the N-terminal segment of receptor, the newly exposed N-terrninal peptide segment binds to the receptor itself as a tethered ligand which activates the receptor [1]. Synthetic heptapeptide Ser-Phe-Leu-Leu-Arg-Asn-Pro (SFLLRNP, one letter amino acid codes) corresponding to this tethered-ligand is able to activate the receptor without thrombin, and the Phe-2 residue of this SFLLRNP peptide was found to be essential for receptor recognition and activation. In the present structure-activity studies to elucidate the role of Phe-2 in receptor activation, the benzene hydrogens in the Phe-2-phenyl group were suggested to be in the edge-to-face CH/π interaction with the receptor aromatic groups. Computer modeling of thrombin receptor indicated that the aromatic amino acid cluster in the fifth transmembrane domain (TM5: YYAYYFSAFSAVFFF) is a binding site of this Phe-2-phenyl. In this study, in order to determine the genuine binding site of Phe-2-phenyl, we prepared mutant receptors in which Tyr in TM5 were replaced by Ala.