Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes

Arch Biochem Biophys. 2001 Feb 15;386(2):195-204. doi: 10.1006/abbi.2000.2207.


Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism
  • Blood Platelets / physiology
  • Calcium / metabolism
  • Calcium Signaling / drug effects
  • Cell Line
  • Directed Molecular Evolution
  • Dose-Response Relationship, Drug
  • Drug Design
  • Humans
  • Ligands
  • Mice
  • Peptide Library
  • Peptides / chemistry*
  • Peptides / genetics
  • Peptides / metabolism
  • Peptides / pharmacology*
  • Platelet Aggregation / drug effects
  • Rats
  • Receptor, PAR-1
  • Receptor, PAR-2
  • Receptors, Thrombin / agonists*
  • Receptors, Thrombin / chemistry
  • Receptors, Thrombin / genetics
  • Receptors, Thrombin / metabolism*
  • Structure-Activity Relationship


  • Ligands
  • Peptide Library
  • Peptides
  • Receptor, PAR-1
  • Receptor, PAR-2
  • Receptors, Thrombin
  • Calcium