Structure-function analysis of protease-activated receptor 4 tethered ligand peptides - Determinants of specificity and utility in assays of receptorfunction

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signal ing. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGRF, lacks poten cy and is of limited utility. In a structure-activity analysis of PARI pept ides, AYPGKF was similar to 10-fold more potent than GYPGKF and, unlike GYP GKF, elicited PAR4-mediated responses comparable in magnitude to those elic ited by thrombin, AYPGKF was relatively specific for PARA in part due to th e tyrosine at position 2; substitution of phenylalanine or p-fluorophenylal anine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors, Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 functi on. For example, AYPGKF activated and desensitized PARA in platelets and, l ike thrombin, triggered phosphoinositide hydrolysis but not inhibition of a denylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR 1, PARA couples to G(q) and not G(i).