Protease-activated receptors start a family.

Cells sense their environment by using specific receptors to detect extracellular ligands. Classically, information transfer occurs by the reversible binding of ligand to receptor. An exception to such reversible ligand-receptor interactions was uncovered with the cloning of a cell surface receptor that mediates responses to thrombin, a serine protease with a host of actions on platelets and other cells. The thrombin receptor is a seven-transmembrane-domain G protein-coupled receptor (1, 2), but it is activated by an unusual mechanism. Thrombin cleaves the amino-terminal exodomain of the receptor. This irreversible proteolytic event unmasks a new amino terminus that serves as a tethered peptide ligand, binding intramolecularly to other receptor domains to effect receptor activation (Fig. 1) (1, 3). It was hoped that the thrombin receptor might sire a family of receptors that use this mechanism, but until now it has remained solitary. This isolation now appears over. In this issue, Nystedt et al. (4) report the cloning of a new proteaseactivated receptor that is closely related to the thrombin receptor. In celebration of this new arrival, dubbed PAR-2 for proteinase-activated receptor 2, this commentary reviews the evidence for the mechanism of receptor activation that defines this fledgling family and notes some of the questions and opportunities raised by it. The distinctive proteolytic mechanism by which thrombin-receptor activation proceeds is relatively well-established (Fig. 1) (1-3, 5, 6). Thrombin recognizes the amino-terminal exodomain of its receptor via two interactions: the LDPR amino acid sequence docks in the active center of thrombin, and the DKYEPF sequence binds the thrombin anionbinding exosite. Thrombin then cleaves the peptide bond between receptor residues Arg-41 and Ser-42 to unmask a new amino terminus beginning with the sequence SFLLRN. Mutation of the Arg41/Ser-42 cleavage site to an "uncleavable" Arg/Pro site rendered the receptor unactivatable by thrombin (1). Replacement of the thrombin-cleavage recognition sequence LDPR/S with DDDDK/S, the recognition site for enteropeptidase, switched receptor specificity. Cells expressing this construct responded to enteropeptidase but did not respond to thrombin (5, 7). Thrombin cleavage of its receptor on intact cells was demonstrated by using antibodies to the activation peptide of the receptor (the fragment cleaved from the receptor by thrombin) vs. other receptor domains retained after cleavage (8). Mutation of the Arg-41/ Ser-42 site to Arg-41/Pro-42 prevented receptor cleavage in these studies. The rates of receptor cleavage and secondmessenger generation correlated well (8). Taken together, these data strongly suggest that cleavage of the Arg-41/Ser-42 peptide bond is not only necessary but sufficient for receptor activation by proteases. The importance of the receptor's DKYEPF sequence for thrombin-receptor interaction has been demonstrated in functional studies with mutant receptors and in biochemical studies with receptorbased peptides (5, 6, 9). These studies identified receptor residues Tyr-52, Glu53, and Phe-55 as key for interaction with thrombin and suggested that they might dock with the anion-binding exosite of thrombin in a manner similar to residues Phe-56, Glu-57, and Ile-59 of the leech anticoagulant hirudin (10) (Fig. 1). Recent x-ray crystallographic studies of cocrystals of thrombin with receptorbased peptides confirmed this analogy (11). The thrombin receptor has thus evolved at least two distinct domains (the LDPR and DKYEPF amino acid sequences) to mediate recognition by thrombin, presumably a device for promoting specificity. How might proteolysis within the amino-terminal extension of a receptor activate that receptor? Synthetic peptides that mimic the new amino terminus created when thrombin cleaves its receptor were full agonists for receptor activation and bypassed the requirement for receptor proteolysis (1, 12, 13). This key observation suggested two possible models (1, 3): (i) the tethered-ligand mechanism described above, and (ii) the thrombin receptor is tonically constrained in an off state by the amino-terminal exodomain; receptor cleavage or competition by exogenous peptide then releases the receptor from this tonic inhibition. This second model was refuted by the observation that a mutant thrombin receptor lacking an amino-terminal exodomain was not constitutively active, as would be predicted by the release-from-inhibition hypothesis, and responded to synthetic peptide representing the tethered-liganddomain peptide like the wild-type receptor. Other experiments confirmed that intra as opposed to intermolecular liganding is the predominant mode of thrombinreceptor activation (3). The thrombin receptor can thus be viewed as a peptide receptor that contains its own agonist. This "agonistpeptide" or "tethered-ligand" domain is kept silent in the naive receptor to be unveiled by receptor cleavage. How is this accomplished? Structure-activity studies with synthetic peptides representing the tethered-ligand domain revealed the peptide's protonated amino group to be one of several features critical for agonist function (12-14). Creation of the corresponding protonated amino group in the receptor by thrombin cleavage of the Arg-41//Ser-42 peptide bond may thus be an important mechanism for "switching on" the intrinsic agonist peptide domain of the receptor. Release from steric interference provided by the activation peptide presumably also contributes. PAR-2 now arrives on the scene, cloned from a mouse genomic library with oligonucleotide probes based on the bovine substance K receptor. The open reading frame encoded a 395-amino acid protein with seven putative transmembrane domains and other features common to G protein-coupled receptors. Search of the European Molecular Biology Laboratory data base revealed PAR-2 to be most closely related to the thrombin receptor with =30% overall amino acid-sequence identity, 42% in the region bounded by transmembrane domains one and seven. This result raised the possibility that PAR-2 might be a protease-activated receptor, and indeed, trypsin proved to be a potent activator of PAR-2 expressed in Xenopus oocytes. Sequence alignment with the thrombin receptor (Fig. 1) suggested a possible cleavage site analogous to that used for activation of the thrombin receptor, and mutation of this site from Arg/Ser to Arg/Pro prevented PAR-2 activation by trypsin. As with the thrombin receptor, a synthetic hexapeptide peptide (SLIGRL) representing the first six amino acids following this putative PAR-2 cleavage