The role of arginine in thrombin receptor tethered-ligand peptide in intramolecular receptor binding and self-activation

Synthetic heptapeptide of the human thrombin receptor tethered-ligand peptide, H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-NH2 (SFLLRNP), activates fully the thrombin receptor without thrombin. The functional role of Arg-5 was examined using a series of analogs having amino acid substitutions at position 5 in this assays was to assess the abilities to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells and to aggregate the human platelet. The replacement of Arg-5 by Ala reduced the activity (9% activity of the parent peptide) in the PI-turnover assay, and abolished completely the platelet aggregation activity. SFLL/Lys/NP was also active, but moderately. 36% in PI-turnover and 12% in platelet aggregation. These results indicated that the electrostatic interaction of the Arg-guanidino group is important for a peptide to interact with the receptor. When citrulline or glutamine was placed at position 5 instead of arginine, the resulting SFLL/citrulline/NP and SFLL/Gln/NP were found to be potent in both assays. Since citrulline and glutamine possess a side chain which can serve as hydrogen donor and/or acceptor, the receptor activation of these peptides appears to be due to hydrogen bonding at this position. The molecular mechanisms to explain both electrostatic and hydrogen-bonding interactions were postulated based on the structural modeling of seven-transmembrane domain thrombin receptor.