Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides

Abstract

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 signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structureactivity analysis of PAR4 peptides, AYPGKF was ϳ10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine 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 AY-PGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G q and not G i .The coagulation protease thrombin elicits biological responses at least in part via G protein-coupled protease-activated receptors (PARs) 1 (1-5). PAR1, the prototype for this family, is activated when thrombin cleaves its amino-terminal exodomain at the R41/S42 peptide bond (1). This serves to unmask a new amino terminus beginning with the sequence SFLLRN, which serves as a tethered peptide agonist, binding intramolecularly to the body of the receptor to effect transmembrane signaling. PARs are thus in essence peptide receptors that carry their own ligands, which remain silent until unmasked by site-specific receptor cleavage. Synthetic peptides that mimic these tethered ligands function as PAR agonists and activate their receptors independent of protease and receptor cleavage (1, 4 -6). Such peptides have been extremely useful for probing the roles of PARs in cells and tissues.PAR4 is a recently characterized thrombin receptor that is expressed in human platelets along with PAR1 (4, 5, 7). Human PAR4 is activated when thrombin cleaves its amino-terminal exodomain at the Arg-47/Gly-48 peptide bond to unmask the tethered ligand GYPGQV (4, 5). The synthetic peptide GYPGQV does function as an agonist for PAR4, but a concentration of 200 -500 M is required for activity. This lack of potency severely limits the utility of this peptide for probing PAR4 function in culture systems and virtually precludes its use in vivo. Structure-function relationships for the PAR4 tethered ligand have not been explored. We here report such a study. In addition to identifying requirements for agonist activity and specificity, this study provides a new PAR4 specific agonist peptide, AY...

Platelet PAR1 receptor density—Correlation to platelet activation response and changes in exposure after platelet activation

“…The new N-terminus formed by thrombin cleavage serves as a "tethered ligand" [4] which binds intramolecularly and causes receptor activation. Specific agonist peptides, mimicking the new N-terminus, have been designed, for PAR1 the most often used is SFLLRN (PAR1-AP) [5], for PAR4 the most potent is AYPGKF (PAR4-AP) [6]. If both cleavage sites are blocked by antibodies raised against peptides spanning the cleavage sites, the platelet response to thrombin is abolished [7].…”

Protease activated receptors: theme and variations

“…Early studies of PAR1 used agonist peptides that were later shown to activate PAR2 (Blackhart et al, 1996;Hollenberg et al, 1997). Over time, relatively speci®c agonist peptides have been described for PAR1, PAR2, and PAR4 (Table 1) (Blackhart et al, 1996;Xu et al, 1998;Faruqi et al, 2000;Hollenberg et al, 1997;Brass et al, 1992). In some cells, one can now tease out contributions from individual PAR subtypes, and con®rm distribution data with functional correlates of receptor expression.…”

“…Granzyme A (Suidan et al, 1994(Suidan et al, , 1996. c Structure/activity relationships have been explored in some detail for short peptides (up to 14 amino acids) that activate PAR1 and PAR2 Scarborough et al, 1992;Al-Ani et al, 1999;Hollenberg et al, 1992Hollenberg et al, , 1997Blackhart et al, 1996;Chao et al, 1992;Natarajan et al, 1995;Kawabata et al, 1999), and more recently for PAR4 (Kahn et al, 1998b(Kahn et al, , 1999Hollenberg et al, 1999;Faruqi et al, 2000). To date, no peptide activators of PAR3 have been described (Hollenberg, 1999).…”

“…In some cells, one can now tease out contributions from individual PAR subtypes, and con®rm distribution data with functional correlates of receptor expression. For example, several recent reports have used agonist peptides to con®rm PAR4 expression in platelets (Xu et al, 1998;Kahn et al, 1998b;Shapiro et al, 2000;Faruqi et al, 2000) as well as in gastric and vascular smooth muscle cells . Still, responses to these agonists (even in combination) may not accurately recreate responses to proteases, and in cells that express PAR3 along with PAR1 or PAR4 it is still necessary to use thrombin.…”

Thrombin‐induced platelet endostatin release is blocked by a proteinase activated receptor‐4 (PAR4) antagonist

“…GYPGKF represents the ®rst six amino acids of the new amino terminus unmasked when thrombin cleaves murine PAR4. A selective PAR4 agonist, AY-NH 2 , is 10 times more potent in activating platelets than a peptide with the sequence of the natural amino terminus (Faruqi et al, 2000;Hollenberg & Saifeddine, 2001). Recently, a selective PAR4 antagonist (tcY-NH 2 ) was described which inhibits thrombin-and AY-NH 2 -induced rat platelet aggregation (Hollenberg & Saifeddine, 2001).…”