Oncogene (2001) 20, 1570 ± 1581 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Protease activated receptors: theme and variations Peter J O'Brien1,2,3,5, Marina Molino4, Mark Kahn1,2,3,6 and Lawrence F Brass*,1,2,3,7 1Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104, USA; 2Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104, USA; 3Center for Experimental Therapeutics at the University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104, USA; 4Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy The four PAR family members are G protein coupled bers, and eorts to identify additional, biologically- receptors that are normally activated by proteolytic relevant proteases that can activate PAR family exposure of an occult tethered ligand. Three of the members. family members are thrombin receptors. The fourth (PAR2) is not activated by thrombin, but can be activated by other proteases, including trypsin, tryptase The current PAR family members and Factor Xa. This review focuses on recent informa- tion about the manner in which signaling through these Four PAR family members have been identi®ed to receptors is initiated and terminated, including evidence date. Three (PAR1, PAR3 and PAR4) are thrombin for inter- as well as intramolecular modes of activation, receptors. The fourth, PAR2, is activated by serine and continuing eorts to identify additional, biologically- proteases other than thrombin (Table 1). PAR1 was relevant proteases that can activate PAR family initially identi®ed using RNA derived from thrombin- members. Oncogene (2001) 20, 1570 ± 1581. responsive cells and of the four current family members it is still the one about which there is the Keywords: protease-activated receptors; thrombin; most known (Rasmussen et al., 1991; Vu et al., platelets; endothelial cells; G proteins; G protein 1991a). It is also the one that established the coupled receptors activation paradigm that in general terms applies to the other three family members (Vu et al., 1991a). That paradigm includes the ability of these receptors Introduction to respond not only to selected proteases, but also to peptides based upon the sequence of the tethered Ten years after the identi®cation of the ®rst protease- ligand. The one exception to the rule is PAR3 for activated G protein coupled receptor, now known as which no activating peptide agonist has been dis- PAR1, the basic principles of its mechanism of covered. In contrast to PAR1, PAR2 was cloned activation have become familiar. Unlike most G serendipitously and only subsequently found to be protein coupled receptors, PAR1 has an intrinsic activated by trypsin (Nystedt et al., 1994, 1995a,b). ligand within its extended N-terminus that is exposed PAR3 was identi®ed as a second thrombin receptor proteolytically by thrombin, creating a neo-N-terminus when gene ablation studies showed that platelets from that serves as a tethered ligand for the receptor (Figure mice lacking PAR1 were still responsive to thrombin 1). The extracellular domains of the receptor form the (Connolly et al., 1996). It is a major regulator of ligand binding site, presumably triggering a conforma- thrombin responses in rodent platelets (Ishihara et al., tional change in the receptor that in turn leads to 1997), but little else is known about it. When over- guanine nucleotide exchange on associated G proteins expressed, human PAR3 can respond to thrombin by and initiates intracellular signaling. Since this basic a mechanism that was initially thought to resemble mechanism has been well covered elsewhere (Grand et PAR1. However, recent evidence suggests that on al., 1996; De ry et al., 1998; Coughlin, 2000), this review murine platelets PAR3 serves solely to facilitate the will primarily focus on recent discoveries concerning cleavage of PAR4 at low thrombin concentrations and receptor activation and inactivation, the evidence for does not directly activate G proteins (Nakanishi- intermolecular relationships among PAR family mem- Matsui et al., 2000). The fourth family member, PAR4, was identi®ed by database searches using conserved domains of the other three family members (Kahn et al., 1998b; Xu et al., 1998). PAR4 is *Correspondence: LF Brass, University of Pennsylvania, Room 913 BRB-II, 421 Curie Blvd., Philadelphia, PA 19104, USA expressed on human and mouse platelets and Current addresses: 5Department of Genetics, University of presumably accounts for the continued ability of Pennsylvania, ARC 509, 3516 Civic Center Blvd., Philadelphia PA platelets from PAR3 knockout mice to respond to 19104, USA; 6Department of Medicine, University of Pennsylvania, thrombin (Kahn et al., 1998b; Xu et al., 1998). 952 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6069, USA; Thus, the four PAR family members share a number 7Departments of Medicine and Pharmacology, Center for Experimental Therapeutics, 913 BRB II/III, 421 Curie Blvd, of similarities, but some dierences have emerged. As Philadelphia, PA 19104-6069, USA noted above, three of the receptors can be activated by Protease-activated receptors PJ O'Brien et al 1571 Figure 1 Structure and features of PAR1. Cartoon of human PAR1 highlighting domains thought to be involved in receptor activation and interactions with thrombin. Additional details and references are included in the text. Potential sites for N-linked glycosylation are marked with an asterisk. L258P, D199R.R199D and Y371A/Y372A/Y373A are some of the known induced mutations that will inactivate PAR1. There are no known naturally occurring PAR1 mutants that result in receptor inactivation thrombin. Two of these, PAR1 and PAR3, have PAR1, PAR2 and PAR3 are in a cluster that maps to similar dose/response curves when studied in expres- 13d2 and PAR4 is located at 8b3 (Kahn et al., 1998a). sion systems. The third, PAR4, requires 10 ± 100-fold If genes for additional PAR family members exist, they higher concentrations of thrombin, apparently because have not yet been identi®ed. it lacks the hirudin-like sequences that can interact Information about the tissue distribution of the four with thrombin's anion-binding exosite and facilitate PAR family members has been obtained by a number receptor cleavage (Kahn et al., 1998b; Xu et al., 1998; of approaches and is most complete for PAR1 and Nakanishi-Matsui et al., 2000). PAR2, since antibodies for these receptors are widely available. These receptors are found in a variety of tissues and cell lines, and have been described in PAR genetics and tissue distribution several species and during embryonic development The genes encoding all four family members have a (recently reviewed in De ry et al. (1998), see also similar structure with a single intron interrupting the Jenkins et al. (2000)). The patterns of expression of sequence encoding the receptor N-terminus. Similarities PAR3 and PAR4 are less well characterized, and rely between PARs extend to their genetic loci, which are primarily upon analyses of mRNA expression. Inter- conserved across species, and suggest a common estingly, while human PAR3 is widely and abundantly ancestral origin (Kahn et al., 1998a). In humans expressed, mouse PAR3 is highly expressed only in PAR1, PAR2, and PAR3 are tightly clustered at megakaryocytes, bone marrow, and spleen (Ishihara et chromosome 5q13, while PAR4 is located separately al., 1997). Human PAR4 mRNA is abundant in at 19p12 (Xu et al., 1998; Kahn et al., 1998a). In mice gastrointestinal tissues including the liver, small Oncogene Protease-activated receptors PJ O'Brien et al 1572 Table 1 PAR family members PAR-1 PAR-2 PAR-3 PAR-4 Activating proteases Thrombin Trypsin Thrombinf Thrombin Trypsin Tryptase Trypsin Xa Xa Cathepsin Gf Granzyme Ab TF/VIIad Cleavage sequence LDPR/SFLLR SKGR/SLIGK LPIK/TFRGAP PAPR/GYPGQV and tethered ligand Protease *EC50 Thrombin 50 pM Trypsin 1 nM Thrombin 0.2 nM Thrombin 5 nM Tryptase 1 nM Trypsin 5 nM Examples of peptide SFLLRN, TFLLRN SLIGKV None known GYPGKF agonistsc GFIYF SFLLRN AYPGKF (Faruqi et al., 2000) Peptide *EC50 SFLLRN 0.1 mM SLIGKV 1 mM ± GYPGQV 100 mM TFLLRN 0.1 mM SFLLRN 1 ± 10 mM Inactivating proteases Plasmin ± ± ± Cathepsin G Elastase Proteinase 3e Examples of antagonists g ±±± Gene deletion in mice Partial embryonic Mild impairment of Loss of thrombin Not yet reported lethality; no hemostatic leukocyte migration; signaling in platelets abnormalities in no embryonic loss at low thrombin surviving knockout (Jia et al., 1999) concentrations; no mice (Connolly et al., 1996; embryonic loss Darrow et al., 1996) (Kahn et al., 1998b) Chromosome 5q13 5q13 5q13 19p12 aThe references included in this table are not intended to be exhaustive. See text as well. bActivating proteases for PAR1: Thrombin (Vu et al., 1991a; Rasmussen et al., 1991). Trypsin (Vouret-Craviari et al., 1995b; Xu et al., 1998; Vu et al., 1991a). Factor Xa (Molino et al., 1997c; Riewald et al., 2000). Granzyme A (Suidan et al., 1994, 1996). cStructure/activity relationships have been explored in some detail for short peptides (up to 14 amino acids) that activate PAR1 and PAR2 (Vassallo et al., 1992; Scarborough et al., 1992; Al-Ani et al., 1999; Hollenberg et al., 1992, 1997; Blackhart et al., 1996; Chao et al., 1992; Natarajan et al., 1995; Kawabata et al., 1999), and more recently for PAR4 (Kahn et al., 1998b, 1999; Hollenberg et al., 1999; Faruqi et al., 2000). To date, no peptide activators of PAR3 have been described (Hollenberg, 1999). dOptimal activation of PAR2 by factor VIIa appears to require co-expression of tissue factor (TF) and the presence of factor X (Camerer et al., 2000). Under these conditions the concentration of factor VIIa that is required to elicit signaling thru PAR2 drops to 8 pM (EC50). This basic phenomenon was obnserved in several mammalian cell lines, umbilical vein endothelial cells and injected Xenopus laevis ocytes (see references below, although not in baby hamster kidney (BHK) cells (Peterson et al., 2000).