Different Roles of Two Consecutive Leucine Residues in a Receptor-Tethered Ligand Peptide (SFLLRNP) in Thrombin Receptor Activation

Abstract

Abstract A synthetic peptide, H–Ser–Phe–Leu–Leu–Arg–Asn–Pro–NH2, which corresponds to a ligand peptide tethered to a human thrombin receptor, was able to activate the thrombin receptor with no thrombin. In order to inspect the structural requisites of two consecutive leucines (Leu-3 and Leu-4) in receptor activation, two sets of analogs with substitutions at either position 3 or 4 were synthesized and evaluated for their ability to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells. The replacement of Leu-4 by Ala drastically decreased the activity of the parent peptide (only about 4% activity), while that of Leu-3 retained about 50% activity. A similar result was obtained when replaced by Gly. Substitution by Phe for Leu-3 sustained full activity. Although the Leu-4/Phe substitution exhibited a slight reduction in activity, Leu-4/Ile substitution unexpectedly diminished the activity (20%). These results suggested that two consecutive leucines have different roles in receptor activation; i.e., Leu-3 behaves something like a connection of peptide units to construct a bioactive conformation and Leu-4 acts like a structural element essential for interactions with receptors.