DRUG DEVELOPMENT RESEARCH 59:367–374 (2003) DDR

Research Overview Intracellular Signalling by the G-Protein Coupled Proteinase-Activated (PAR) Family Scott R. Macfarlane and Robin Plevinn Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, United Kingdom

Strategy, Management and Health Policy

Preclinical Development Clinical Development Venture Capital Preclinical Toxicology, Formulation Phases I-III Postmarketing Enabling Research Drug Delivery, Regulatory, Quality, Phase IV Technology Pharmacokinetics Manufacturing

ABSTRACT The proteinase-activated receptor (PAR) family are novel members of the G-protein coupled receptor superfamily that are activated by a mechanism involving specific proteolytic cleavage of their N-terminal. Activation of intracellular signalling pathways by the PAR family has received relatively little attention compared with the other aspects of their function. Along with activation of intracellular calcium, diacylglycerol, and inositol 1,4,5-trisphosphate, PARs have been reported to activate signalling pathways closely linked to both cell proliferation and inflammation. Here we attempt to describe these signalling pathways and the G-proteins involved in coupling the receptors to these intracellular events. Drug Dev. Res. 59:367–374, 2003. c 2003 Wiley-Liss, Inc.

Key words: PAR; G-protein coupled receptor signalling; MAP kinase; mitogenesis; inflammation

INTRODUCTION receptors have been less fully investigated, and are In addition to the known role for thrombin in the only now beginning to be unraveled. These signalling blood-clotting cascade, various studies reported the pathways provide the framework for the effects of enzyme had specific cellular effects [Chen and receptor activation at a cellular level, and therefore Buchanan, 1975; Bar-Shavit et al., 1983], the most understanding them is of considerable importance if prominent of which was the induction of cellular the PAR family is to be viewed as important proliferation. The mechanism by which thrombin therapeutic targets. In this report, we discuss the brought about these cellular effects was unknown; intracellular signalling pathways identified as being however, a sea change in the physiological role of activated by the PAR family, and attempt to link the thrombin occurred in 1991 with the cloning of a novel activation of these pathways with the known function of G-protein coupled receptor (GPCR), designated pro- the PARs in various tissue types. tease-activated receptor-1 or PAR1. This new receptor was found to be activated by thrombin-mediated cleavage of the receptor protein and to have a wider Grant sponsor: Kowa Company, Japan; Grant sponsor: range of expression than solely cell types involved in Medical Research Council; Grant sponsor: Biotechnology and Biological Sciences Research Council. blood clotting [Vu et al., 1991]. Major advances have n been made in our understanding of the pathophysio- Correspondence to: Prof. Robin Plevin, Dept. Physiology and Pharmacology, SIBS, University of Strathclyde, 27 Taylor logical roles played by this novel family of G-protein Street, Glasgow. G4 0NR, UK. coupled receptors since the discovery of the PAR1 E-mail: [email protected] receptor. However, insights into the intracellular Published online in Wiley InterScience (www.interscience. signalling events that follow activation of these wiley.com) DOI: 10.1002/ddr.10305

c 2003 Wiley-Liss, Inc. 368 MACFARLANE AND PLEVIN

PAR1 AND G-PROTEIN INTERACTIONS [Offermanns et al., 1994], these findings demonstrate that PAR1 can activate a wide range of intracellular The prototypic member of that PAR family, PAR1, signalling pathways including the p42/44 MAP kinase has been the most widely studied of this family, not via activation of c-Src, JNK and PI-3 kinase [Mitchell et only in terms of receptor’s general physiological role, al., 1990; Walker et al., 1998; Swift et al., 2000] (Fig. 1). but also with respect to intracellular signalling. Traditionally it was believed that the a subunits of Association of this seven transmembrane domain- the G-protein family were predominant in the trans- receptor with heterotrimeric G-proteins has become duction of signals from receptors. However, a role for well established, and PAR1 has also been identified as the bg-subunits has been demonstrated and has been influencing a wide range of intracellular signalling recently proposed in the signalling of PAR1 to a intermediates [Macfarlane et al., 2001]. number of signalling pathways including PI-3K (see G-proteins transduce signals to cells from recep- below) and the inflammatory nuclear factor kB (NFkB) tors by coupling the receptors to a number of effector transcription factor signalling pathway [Rahman et al., enzymes. Activation of these effector enzymes, such as 2002]. This transcription factor has previously been adenylyl cyclase, allows the receptors a wide sphere of identified as being activated by PAR1 [Nakajima et al., influence within the cell by changing the activities in 1994; Bretschneider et al., 1997]. This more recent various intracellular signalling cascades. G-proteins fall study adds some detail to the upstream signalling into three groups of subunits, Ga,Gb, and Gg and pathway utilized in the activation of this pathway. their importance to intracellular signalling from seven Another aspect of Ga subunit function is the activation transmembrane domain receptors has become an of small molecular weight G-proteins such as Rac, Ras, important area of study within pharmacology [Neves and Rho [Matozaki et al., 2000]. The activation of et al., 2002]. Included amongst the G-protein coupled members of this superfamily has been identified for receptor family are a- and b-adrenoceptors, muscarinic PAR1 with the identification of Rho activation via PTX- cholinoceptors, and 5-HT receptors amongst others insensitive G12/G13, a mechanism implicated in regula- [Albert and Robillard, 2002]. This highlights the wide tion of cellular invasion in cancer [Nguyen et al., 2002]. reliance and importance of this multifunctional form of signal transduction as these receptors often control PAR1 COUPLING TO P42/44 MAP KINASE vital cellular processes. G-protein a subunits are ACTIVATION separated into four main subfamilies Gs,Gi,Gq, and Activation of the MAP kinase family of proteins G12, with 27 encoding the various members of by PAR1 has received much attention [Van Corven the a subunit families, 5 genes encoding b subunits, et al., 1993; Chen et al., 1996; Apostolidis and Weiss, and a further 14 g genes, and there is a wide scope for 1997], with strong evidence for the coupling of PAR1 to diverse heterotrimeric complexes to form [Albert and the Raf/MEK system of p42/44 activation via the Gi Robillard, 2002] and, therefore, many possibilities for family. The activation of these important cellular interactions with signalling pathways. kinases is well established for tyrosine kinase-linked PAR1 was initially demonstrated to bring about receptors [Malarkey et al., 1995] and signalling two main signalling events. Firstly inhibition of paradigms for the linkage of G-protein coupled adenylyl cyclase and subsequent inhibition of cAMP receptors to the MAP kinases have also been estab- production through interaction with inhibitory mem- lished. However, PAR1 has been shown to use both bers of the Ga family [Hung et al., 1992; Kanthou activation of this pathway via Src and also by et al., 1996] and, secondly, stimulation of phospholipase transactivation of the pathway via growth factor C (PLC) initiated inositol 1,4,5-trisphosphate produc- receptor kinases (see below) [Sabri et al., 2002]. tion (IP3) [Babich et al., 1990; Hung et al., 1992]. Additionally, a role for the PI-3 kinase pathway has Through this pathway, the receptor can bring about been identified in the activation of p42/44 MAP kinase both intracellular calcium release, via the action of IP3 [Malarkey et al., 1995; Touhara et al., 1995]. In addition on inositol 1,4,5-trisphosphate receptors the endoplas- to utilising the tyrosine kinase receptor associated mic reticulum (ER), and activation of members of the protein isoform of PI-3 kinase p85, thrombin also PKC superfamily through the generation of the activates the p110 kDa isoform that is directly activated endogenous PKC activator, diacylglycerol (DAG). by G-protein bg subunits. Furthermore, the activation Subsequently, more detailed studies have identified of PI-3 kinase signalling by thrombin has been roles for Gq/11,Gi2, and Go subunits in PAR1 mediated implicated in downstream activation of important signalling [Babich et al., 1990; Brass et al., 1991; Baffy mitogenic and cell survival proteins such as p70s6k et al., 1994; Ogino et al., 1996]. Taken together with and protein kinase B [Belham et al., 1997; Krymskaya reported effects via the G12/13 class of a subunit et al., 1999; Walker et al., 1998]. The potential PAR-MEDIATED SIGNAL TRANSDUCTION 369 importance of PAR1 as a regulator of cellular prolifera- often observed to thrombin [Kahan et al., 1992]. It is, tion is therefore further highlighted by the activation of therefore, important that thrombin and PAR1 peptide these mitogenic proteins. responses are reassessed in order to establish which Transactivation has become an important concept members of the PAR family are responsible for the in the activation of signalling pathways by G-protein discrepant responses to the two agonists. coupled receptors [Daub et al., 1997; Maudsley et al., 2000]. This mechanism enables GPCRs to activate a PAR2 SIGNALLING cellular kinase that phosphorylates and activates a The discovery of PAR2 underlined the potential growth factor receptor, therefore allowing the GPCR to importance of serine proteinase-mediated receptor utilise the growth factor receptor signalling machinery. activation. Tissue distribution of PAR2 has proved to Research to date suggests that PAR1 uses this strategy be fairly wide, indicating that it may have a role in not only to activate p42/44 MAP kinase [Weiss and several physiological systems and classical pharmaco- Maduri, 1993; Delafontaine et al., 1996], but also in the logical studies have supported PAR2 as having a role in activation of the stress-activated protein kinase p38 many different tissue types [Macfarlane et al., 2001]. At MAP kinase [Kanda et al., 2001]. It should be noted, a cellular level, there are still relatively few studies however, that the use of transactivation to activate assessing the signalling pathways activated by PAR2, p42/44 MAP kinase might be a cell-type specific event possibly due to relatively low expression levels of this [Sabri et al., 2003; Wang et al., 2002]. In general, it is receptor. PAR2 has been closely linked with both cell possible that transactivation pathways activated via proliferation [Mirza et al., 1996; Akers et al., 2000; G-protein coupled receptors represent late phase Frungieri et al., 2002; Gaca et al., 2002] and activation of the signalling pathways, allowing long- inflammation [Wakita et al., 1997; Vergnolle, 1999, term signals to be maintained. 2000; Cocks and Moffat 2000; Vergnolle et al., 2001; PAR1 is a well-established activator of multiple Temkin et al., 2002; Ferral et al., 2003] in many tissue signalling pathways within cells, with many of these types and the signalling pathways thus far identified as pathways directly involved in cell growth and differ- being activated by PAR2 are typically associated with entiation. However, there is increasing evidence for these proliferative and inflammatory cellular processes. PAR1 activation of the inflammatory transcription It has been established that PAR2 activation by factor NFkB [Nakajima et al., 1994; Bretschneider trypsin or agonist peptide leads to increases in et al., 1997; Rahman et al., 2002]. The actions of both intracellular calcium levels together with the produc- thrombin and PAR1 agonist peptide have been linked tion of IP3 and DAG [Nystedt et al., 1995; Santulli to cell proliferation in vascular smooth muscle [Naka- et al., 1995]. This signalling profile, typical of Gq/G11 jima et al., 1994; Bretschneider et al., 1997], and PAR1 coupled receptors acting via PLC isoforms, allows the also increases the expression of adhesion molecules in receptor to influence a wide range of intracellular vascular endothelial cells via a pathway involving NFkB targets, similar to the case for PAR1. In addition, [Minami et al., 2002]. The exact delineation of the experiments with PAR2 expressed in the Xenopus signal from PAR1 that brings about activation of the oocyte system have indicated a degree of pertussis NFkB dimer is, as yet, unidentified. However, recent toxin sensitivity in PAR2 intracellular signalling events, work has pointed to the involvement of both PI-3 indicating a role for Gi/Go-dependent signalling kinase and PKCd in this event [Minami et al., 2002], [Schultheiss et al., 1997]. However relative to PAR1, although the role of the inhibitory kappaB kinase (IKK) little information is available regarding PAR2 coupling isoforms, found upstream of NFkB, was not assessed. to G-proteins. Furthermore, this study reported the activation of Nevertheless, PAR2 has been strongly linked to PKCz by thrombin, leading to the activation of the activation of the MAP kinase family p42/44 MAP downstream GATA signalling pathways. kinase (ERKs), JNK, and p38 MAP kinase. p42/44 Some discrepancies have been observed between MAP kinases are strongly linked with mitogenesis the signalling pathways activated by PAR1 agonist [Pages et al., 1993] and are ubiquitously expressed in peptides and the enzymatic activator of the receptor, mammalian tissues [Pearson et al., 2001]. This kinase thrombin [Vouret-Craviari et al., 1992]. The reason for cascade probably represents one of the most intensely these divergent responses has become clearer in the studied kinase signalling pathways to date, and has a light of discoveries indicating the existence of other well-defined multi-step format that holds for all thrombin receptors. In particular, the cloning and members of the family (see Fig. 1). The activation of characterisation of PAR4 indicates that this receptor p42/44 MAP kinase by PAR2 has been identified in may control a late-phase of thrombin signalling several cell types [Belham et al., 1996; DeFea et al., corresponding to the latter part of two-phase responses 2000; Sabri et al., 2000; Gaca et al., 2002], and due to 370 MACFARLANE AND PLEVIN 2001; Ducroc et al., 2002; Jin et al., 2003], in addition to the receptor being linked to invasiveness in pancreatic cancer [Ikeda et al., 2003]. It has also been demonstrated that PAR2 can activate the important SH2-containing protein tyrosine phosphatase SHP-2 [Yu et al., 1997] that has previously been implicated in PAR1 activation of mitogenesis. Other tyrosine phos- phorylations observed following activation of PAR1 have yet to be demonstrated for PAR2, despite these events relating to PAR1 activation, and transactivation, of p42/44 MAP kinase pathways. These differences possibly indicate fundamental differences in the signalling strategies employed by these two receptors. The use of tyrosine kinase inhibitors has been a feature of some pharmacological studies [Kawabata et al., 1999; Mule et al., 2002], but due to the fairly non- specific nature of these agents, the exact identity of the kinases involved remains conjectural.

Fig. 1. G-protein dependent mitogenic signalling from PAR1. Dashed PAR2 ACTIVATION OF INFLAMMATORY lines represent putative pathways where intermediates have not been SIGNALLING PATHWAYS fully described for PAR1 signalling. Some well-defined intermediates and precursors have been omitted for the sake of clarity. MAPK, p42/ The coupling of PAR2 with the SAP kinases JNK 44 mitogen-activated protein kinase; PKB, protein kinase B; PKC, and p38 MAP kinase, together with the activation of protein kinase C; PIP3, phosphatidyl 3,4,5-trisphosphate. the NFkB signalling pathway, is consistent with the overall view of PAR2 as being involved in inflammatory events (Fig. 2). Activation of c-fos, AP-1, CREB, and NFkB transcriptional activities by PAR [Yu et al., the strong link between PAR and cell proliferation it 2 2 1997; Kanke et al., 2001; Temkin et al., 2002] also was predictable that this important regulator of cell indicates the receptor is capable of bringing about growth and differentiation would be activated by PAR2. The upstream coupling of PAR2 to these signalling pathways still requires further clarification to fully establish the G-proteins and other signalling inter- mediates involved. It has not, however, been estab- lished if PAR2 utilizes transactivation of growth factor signalling pathways to activate the MAP kinase, or any other, cascades. Interestingly, the activation of p42/44 MAP kinase by PAR2 has been suggested to require the internalization of the receptor [DeFea et al., 2000], indicating that the receptor may use alternative modes of signal transduction than merely directly activating G-protein intermediates. This work also provides an interesting adjunct to the idea of G-protein activation of the p42/44 MAP kinase cascade, as the necessity for receptor internalization may indicate an alternative mechanism for cascade activation. Possibly such a mechanism plays a role to aid in the appropriate subcellular localisation of the MAP kinase [Luttrell, 2002], together with maintaining the signal past the point of desensitization of the receptor. Mitogenic signalling activated via PAR may Fig. 2. PAR2 activated Gq/11 coupled inflammatory signalling path- 2 ways. Dashed lines represent pathways where intermediates have not prove to be an important area of study, due to the been fully described for PAR2 signalling. PLCb, phospholipase Cb; increasing evidence that identifies PAR2 expression as IKK, inhibitory kappaB kinase; JNK, jun N-terminal kinase; PKC, being increased in several tumor types [Darmoul et al., protein kinase; TFs, transcription factors. PAR-MEDIATED SIGNAL TRANSDUCTION 371 meaningful changes in the expression of important intracellular calcium in human astrocytoma cells genes within cells. The SAP kinase and NFkB [Kaufmann et al., 2000]. More recently the suggestion signalling pathways are well-documented regulators of has been made that in cardiomyocytes the PAR4 inflammatory expression, and have been impli- specific agonist peptide AYPGFK can activate p38 cated in the control of cytokines such as IL-6, released MAP kinase, but shows limited ability to activate PLC from keratinocytes following PAR2 activation [Wakita and p42/44 MAP kinase [Sabri et al., 2003]. Interest- et al., 1997], IL-8, and GM-CSF, amongst others. ingly, this study also contends that in these cells the Activation of these inflammation-related signalling activation of c-Src by thrombin is a PAR4 mediated pathways by PAR2 has been demonstrated in diverse event, and that the activation of p38 MAP kinase is via cell types such as cardiomyocytes [Sabri et al., 2000], transactivation of the epidermal growth factor receptor coronary smooth muscle [Bretschneider et al., 1999], (EGFR) signalling intermediates. eosinophils [Temkin et al., 2002], and keratinocytes [Kanke et al., 2001], indicating that the activation of CONCLUDING REMARKS these signalling pathways is an important response to As noted earlier, the scope of the studies into the the activation of PAR2. Indeed, recent work points to a intracellular signalling of the PAR family as a whole has potentially important role for PAR2 in rheumatoid been fairly limited. However, as the pace of research arthritis, a disease typified by chronic inflammation into this novel family of receptors increases as it has in [Ferral et al., 2003]. recent years, it is likely that the methods employed by The signalling of PAR2 to the transcription factor these receptors to signal within cells will come under k NF B also requires further investigation. Previous greater scrutiny. This should lead to better under- study of this cascade has indicated that activation of the standing of the PARs and advance their potential as k NF B dimer may proceed via an IKK isoform- therapeutic targets in a wide range of disease states. independent mechanism [Kanke et al., 2001]. This finding raises the possibility that PAR2 may activate a unique pathway for activation of this important ACKNOWLEDGMENTS inflammatory transcription factor. Such a pathway PAR research work in our laboratory has been may enable PAR2 signals to NFkB to be isolated for funded by MRC (ROPA). treatment of inflammatory conditions in which PAR2 blockade is desirable. REFERENCES

PAR3 AND PAR4 ACTIVATED SIGNALLING Akers IA, Parsons M, Hill R, Hollenberg MD, Sanjar S, Laurent GJ McAnulty RJ. 2000. Mast cell tryptase stimulates human lung The investigation of signalling by PAR3 and PAR4 fibroblast proliferation via protease-activated receptor-2. Am J has received very little attention in the literature. As Physiol-Lung Cell Mol Physiol 278:L193–L201. the roles these receptors play in physiological systems Albert PR, Robillard L. 2002. protein specificity: Traffic direction are elucidated, the intracellular signalling mechanisms required. Cell Signal 14:407–418. associated with them shall become clearer. It has Apostolidis A, Weiss RH. 1997. Divergence in the G-protein- proved particularly difficult to establish the role played coupled receptor mitogenic signalling pathway at the level of Raf kinase. Cell Signal 9:439–445. by PAR3 due to the initial lack of a functional synthetic agonist peptide for the receptor [Ishihara et al., Babich M, King KL, Nissenson RA. 1990. Thrombin stimulates 1997; Nakanishi-Matsui et al., 2000; Sambrano et al., inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells. 2001]. Murine PAR3, in contrast to other members of Endocrinology 126:948–954 the PAR family, does not appear to signal in response to Baffy G, Yang L, Raj S, Manning DR, Williamson JR. 1994. G its synthetic tethered activating peptide sequence, and protein coupling to the thrombin receptor in Chinese hamster unequivocal evidence for a response of human PAR3 to lung fibroblasts. J Biol Chem 269:8483–8487. the synthetic PAR3-derived receptor-activating peptide Bar-Shavit R, Kahn A, Fenton JWD, Wilner GD. 1983. Receptor- has yet to be established. At present, it appears that mediated chemotactic response of a macrophage cell line (J774) PAR3 functions principally as a co-factor for the to thrombin. Lab Invest 49:702–707. activation of PAR4 [Nakanishi-Matsui et al., 2000; Belham CL, Tate RJ, Scott PH, Pemberton AD, Miller HRP, Sambrano et al., 2001]. However, the expression of Wadsworth RM, Gould GW, Plevin R. 1996. Trypsin stimulates PAR and PAR in different tissues, either alone or in proteinase-activated receptor-2-dependent and -independent 3 4 activation of mitogen-activated protein kinases. Biochem J combination suggests that PAR3 may play a functional 320:939–946. role that has yet to be determined. Belham CM, Scott PH, Twomey DP, Gould GW, Wadsworth RM, An early study of PAR4 identified that activation Plevin R. 1997. Evidence that thrombin-stimulated DNA synth- of this receptor can lead to increases in the level of esis in pulmonary arterial fibroblasts involves phosphatidylinositol 372 MACFARLANE AND PLEVIN

3-kinase-dependent p70 ribosomal S6 kinase activation. Cell vated receptor-2 in human pancreatic cancer: A possible relation Signal 9:109–116. to cancer invasion and induction of fibrosis. Int J Oncol 22: Brass LF, Manning DR, Williams AG, Woolkalis MJ, Poncz M. 295–300. 1991. Receptor and -mediated responses to thrombin in Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, HEL cells. J Biol Chem 266:958–965. Timmons C, Tram T, Coughlin SR. 1997. Protease-activated Bretschneider E, Wittpoth M, Weber AA, Glusa E, Schror K. 1997. receptor 3 is a second thrombin receptor in humans. Nature Activation of NFkappaB is essential but not sufficient to stimulate 386:502–506. mitogenesis of vascular smooth muscle cells. Biochem Biophys Jin E, Fujiwara M, Pan X, Ghazizadeh M, Arai S, Ohaki Y, Kajiwara Res Comm 235:365–368. K, Takemura T, Kawanami O. 2003. Protease-activated receptor Bretschneider E, Kaufmann R, Braun M, Wittpoth M, Glusa E, (PAR)1 and PAR2 participate in the cell growth of alveolar Nowak G, Schror K. 1999. Evidence for proteinase-activated capillary endothelium in primary lung adenocarcinomas. Cancer 97:703–713. receptor-2 (PAR2)-mediated mitogenesis in coronary artery smooth muscle cells. Br J Pharmacol 126:1735–1740. Kahan C, Seuwen K, Meloche S, Pouyssegur J. 1992. Coordinate, Chen LB, Buchanan JM. 1975. Mitogenic activity of blood Biphasic activation of p44 mitogen-activated protein-kinase and s6 components. I. Thrombin and prothrombin. Proc Natl Acad Sci kinase by growth-factors in hamster fibroblasts: evidence USA 72:131–135. for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem 267: Chen Y, Grall D, Salcini AE, Pelicci PG, Pouyssegur J, Van 13369–13375. Obberghen-Schilling E. 1996. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein- Kanda Y, Mizuno K, Kuroki Y, Watanabe Y. 2001. Thrombin- coupled thrombin receptor. EMBO 15:1037–1044. induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation Cocks TM, Moffatt JD. 2000. Protease-activated receptors: sentries pathway. Br J Pharmacol 132:1657–1664. for inflammation? Trends Pharmacol Sci 21:103–108. Kanke T, Macfarlane SR, Seatter MJ, Davenport E, Paul A, Darmoul D, Marie JC, Devaud H, Gratio V, Laburthe M. 2001. McKenzie RC, Plevin R. 2001. Proteinase-activated receptor-2- Initiation of human colon cancer cell proliferation by trypsin mediated activation of stress-activated protein kinases and acting at protease-activated receptor-2. Br J Cancer 85:772–779. inhibitory kappa B kinases in NCTC 2544 keratinocytes. J Biol Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A. 1997. Chem 276:31657–31666. Signal characteristics of G protein-transactivated EGF receptor. Kanthou C, Kanse SM, Kakkar VV, Benzakour O. 1996. Involvement EMBO 16:7032–7044. of pertussis toxin-sensitive and -insensitive G proteins in alpha- DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett thrombin signalling on cultured human vascular smooth muscle NW. 2000. beta-Arrestin-dependent endocytosis of proteinase- cells. Cell Signal 8:59–66. activated receptor 2 is required for intracellular targeting of Kaufmann R, Patt S, Zieger M, Kraft R, Tausch S, Henklein P, activated ERK1/2. J Cell Biol 148:1267–1281. Nowak G. 2000. The two-receptor system PAR1/PAR4 mediates Delafontaine P, Anwar A, Lou H, Ku L. 1996. G-protein coupled alpha-thrombin- induced [Ca2+](i) mobilization in human astro- and tyrosine kinase receptors: evidence that activation of the cytoma cells. J Cancer Res Clin Oncol 126:91–94. insulin-like growth factor I receptor is required for thrombin- Kawabata A, Kuroda R, Nishikawa H, Kawai K. 1999. Modulation induced mitogenesis of rat aortic smooth muscle cells. J Clin by protease-activated receptors of the rat duodenal motility in Invest 97:139–145. vitro: possible mechanisms underlying the evoked contraction and Ducroc R, Bontemps C, Marazova K, Devaud H, Darmoul D, relaxation. Br J Pharmacol 128:865–872. Laburthe M. 2002. Trypsin is produced by and activates protease- Krymskaya VP, Penn RB, Orsini MJ, Scott PH, Plevin RJ, activated receptor-2 in human cancer colon cells: evidence for Walker TR, Eszterhas AJ, Amrani Y, Chilvers ER, Panettieri new autocrine loop. Life Sci 70:1359–1367. RA. 1999. Phosphatidylinositol 3-kinase mediates mitogen-in- Ferral WR, Lockhart JC, Kelso EB, Dunning L, Plevin R, Meeks duced human airway smooth muscle cell proliferation. Am J SE, Smith AJH, Hunter GD, McLean JS, McGarry F, Ramage R, Physiol-Lung Cell Mol Physiol 277:L65–L78. Jiang L, Kanke T, Kawagoe J. 2003. Essential role for proteinase- Luttrell LM. 2002. Activation and targeting of mitogen-activated activated receptor-2 in arthritis. J Clin Invest 111:35–41. protein kinases by G-protein-coupled receptors. Can J Physiol Frungieri MB, Weidinger S, Meineke V, Kohn FM, Mayerhofer A. Pharmacol 80:375–382. 2002. Proliferative action of mast-cell tryptase is mediated by Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. 2001. PAR2, COX2, prostaglandins, and PPARgamma: Possible rele- Proteinase-activated receptors. Pharmacol Rev 53:245–282. vance to human fibrotic disorders. Proc Natl Acad Sci USA Malarkey K, Belham CM, Paul A, Graham A, McLees A, Scott PH, 99:15072–15077. Plevin R. 1995. The regulation of tyrosine kinase signalling Gaca MD, Zhou X, Benyon RC. 2002. Regulation of hepatic stellate pathways by growth factor and G-protein-coupled receptors. cell proliferation and collagen synthesis by proteinase-activated Biochem J 309:361–375. receptors. J Hepat 36:362–369. Matozaki T, Nakanishi H, Takai Y. 2000. Small G-protein networks: Hung DT, Wong YH, Vu TK , Coughlin SR. 1992. The cloned Their crosstalk and signal cascades. Cell Signal 12:515–524. platelet thrombin receptor couples to at least two distinct Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, effectors to stimulate phosphoinositide hydrolysis and inhibit Lefkowitz RJ, Luttrell LM. 2000. The beta(2)- adenylyl cyclase. J Biol Chem 267:20831–20834. mediates extracellular signal- regulated kinase activation via Ikeda O, Egami H, Ishiko T, Ishikawa S, Kamohara H, Hidaka H, assembly of a multi-receptor complex with the epidermal growth Mita S, Ogawa M. 2003. Expression of proteinase-acti- factor receptor. J Biol Chem 275: 9572–9580. PAR-MEDIATED SIGNAL TRANSDUCTION 373

Minami T, Abid MR, Zhang J, King G, Kodama T, Aird WC. 2002. Sabri A, Short J, Guo J, Steinberg SF. 2002. Protease-activated Thrombin stimulation of VCAM-1 in endothelial cells is mediated receptor-1-mediated DNA synthesis in cardiac fibroblast is via by PKC-delta-NFkappa B and PKC-zeta-GATA signalling path- epidermal growth factor receptor transactivation: distinct PAR1 ways. J Biol Chem 278:6976–6984. signaling pathways in cardiac fibroblasts and cardiomyocytes. Circ Mirza H, Yatsula V, Bahou WF. 1996. The proteinase activated Res 91:532–539. receptor-2 (PAR2) mediates mitogenic responses in human Sabri A, Guo J, Elouardighi H, Darrow AL, Andrade-Gordon P, vascular endothelial cells: molecular characterization and evi- Steinberg SF. 2003. Mechanisms of protease-activated receptor-4 dence for functional coupling to the thrombin receptor. J Clin actions in cardiomyocytes: role of Src tyrosine kinase. J Biol Chem Invest 97:1705–1714. 278:11714–11720. Mitchell CA, Jefferson AB, Bejeck BE, Brugge JS, Deuel TF, Sambrano GR, Weiss EJ, Zheng YW, Huang W, Coughlin SR. 2001. Majerus PW. 1990. Thrombin-stimulated immunoprecipitation of Role of thrombin signalling in platelets in haemostasis and phosphatidylinositol 3-kinase from human platelets. Proc Nat thrombosis. Nature 413:6851:74–78. Acad Sci USA 87:9396–9400. Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Mule F, Baffi MC, Falzone M, Cerra MC. 2002. Signal trans- Seiberg M, Scarborough RM, Andrade-Gordon P. 1995. Evidence duction pathways involved in the mechanical responses to for the presence of a protease-activated receptor distinct from the protease-activated receptors in rat colon. J Exp Ther 303: thrombin receptor in human keratinocytes. Proc Natl Acad Sci 1265–1272. 92:9151–9155. Nakajima T, Kitajima I, Shin H, Takasaki I, Shigeta K, Abeyama K, Schultheiss M, Neumcke B, Richter HP. 1997. Endogenous trypsin Yamashita Y, Tokioka T, Soejima Y, Maruyama I. 1994. Involve- receptors in Xenopus oocytes: linkage to internal calcium stores. ment of NF-kappa B activation in thrombin-induced human Cell Mol Life Sci 53:842–849. vascular smooth muscle cell proliferation. Biochem Biophys Res Swift S, Sheridan PJ, Covic L, Kuliopulos A. 2000. PAR1 thrombin Commun 204:950–958. receptor-G protein interactions: separation of binding and Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman coupling determinants in the . J Biol Chem MJ, Coughlin SR. 2000. PAR3 is a cofactor for PAR4 activation by 275:2627–2635. thrombin. Nature 404:609–613. Temkin V, Kantor B, Weg V, Hartman ML, Levi-Schaffer F. 2002. Neves SR, Ram PT, Lyengar R. 2002. G protein pathways. Science Tryptase activates the mitogen-activated protein kinase/ 296:1636–1639. activator protein-1 pathway in human peripheral blood eosino- Nguyen QD, Faivre S, Bruyneel E, Rivat C, Seto M, Endo T, phils, causing cytokine production and release. J Immunol Mareel M, Emami S, Gespach C. 2002. RhoA- and RhoD- 169:2662–2669. dependent regulatory switch of Galpha subunit signalling by PAR1 Touhara K, Hawes BE, Vanbiesen T, Lefkowitz RJ. 1995. receptors in cellular invasion. FASEB J 16:565–576. G-protein beta-gamma-subunits stimulate phosphorylation Nystedt S, Emilsson K, Larsson A K, Strombeck B, Sundelin J. of SHC adapter protein. Proc Natl Acad Sci USA 92:9284–9287. 1995. Molecular cloning and functional expression of the gene Van Corven EJ, Hordijk PL, Medema RH, Bos JL, Moolenaar WH. encoding the human proteinase-activated receptor 2. Eur J 1993. Pertussis toxin-sensitive activation of P21(Ras) by G- Biochem 232:84–89. protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Offermanns S, Laugwitz KL, Spicher K, Schultz G. 1994. Sci USA 90:1257–1261. G proteins of the G12 family are activated via thromboxane A2 Vergnolle N. 1999. Proteinase-activated receptor-2-activating pep- and thrombin receptors in human platelets. Proc Nat Acad Sci tides induce leukocyte rolling, adhesion, and extravasation in vivo. 91:504–508. J Immunol 163:5064–5069. Ogino Y, Tanaka K, Shimizu N. 1996. Direct evidence for two Vergnolle N. 2000. Review article: proteinase-activated receptors: distinct G proteins coupling with thrombin receptors in novel signals for gastrointestinal pathophysiology. Aliment Phar- human neuroblastoma SH-EP cells. Eur J Pharmacol 316: macol Ther 14:257–266. 105–109. Vergnolle N, Hollenberg MD, Wallace JL. 2000. Injection of Pages G, Lenormand P, L’Allemain G, Chambard JC, Meloche S, proteinase-activated receptor-2-activating peptides (PAR2-APs) in Pouyssegur J. 1993. Mitogen-activated protein kinases p42mapk the rat hindpaw induces hyperalgesia. FASEB J 14:A386. and p44mapk are required for fibroblast proliferation. Proc Natl Vergnolle N, Wallace JL, Bunnett NW, Hollenberg MD. 2001. Acad Sci USA 90:8319–8323 Protease-activated receptors in inflammation, neuronal signaling Pearson G, Robinson F, Gibson TB, Xu B-E, Karandikar M, Berman and pain. Trends Pharmacol Sci 22:146–152. K, Cobb MH. 2001. Mitogen-activated protein (MAP) kinase Vouret-Craviari V, Van Obberghen-Schilling E, Rasmussen UB, pathways: regulation and physiological functions. Endocr Rev Pavirani A, Lecocq JP, Pouyssegur J. 1992. Synthetic alpha- 22:53–183. thrombin receptor peptides activate G protein-coupled signaling Rahman A, True AL, Anwar KN, Ye RD, Voyo-Yasenetskaya TA, pathways but are unable to induce mitogenesis. Mol Biol Cell Malik AB. 2002. Galpha(q) and Gbetagamma regulate 3:95–102. PAR1 signaling of thrombin-induced NF-kappaB activation Vu TK, Hung DT, Wheaton VI, Coughlin S. 1991. Molecular cloning and ICAM-1 transcription in endothelial cells. Circ Res 91: of a functional thrombin receptor reveals a novel proteolytic 398–405. mechanism of receptor activation. Cell 64:1057–1068. Sabri A, Muske G, Zhang HL, Pak E, Darrow A, Andrade-Gordon P, Wakita H, Furukawa F, Takigawa M. 1997. Thrombin and trypsin Steinberg SF. 2000. Signaling properties and functions of two induce granulocyte-macrophage colony-stimulating factor and distinct cardiomyocyte protease-activated receptors. Circ Res interleukin-6 gene expression in cultured normal human kerati- 86:1054–1061. nocytes. Proc Assoc Am Physicians 109:190–207. 374 MACFARLANE AND PLEVIN

Walker TR, Moore SM, Lawson MF, Panettieri RA Jr, Chilvers ER. Weiss RH, Maduri M. 1993. The mitogenic effect of thrombin in 1998. Platelet-derived growth factor-BB and thrombin activate vascular smooth muscle cells is largely due to basic fibroblast phosphoinositide 3-kinase and protein kinase B: role in growth factor. J Biol Chem 268:5724–5727. mediating airway smooth muscle proliferation. Mol Pharmacol 54: Yu Z, Ahmad S, Schwartz JL, Banville D, Shen SH. 1997. Protein- 1007–1015. tyrosine phosphatase SHP2 is positively linked to proteinase- Wang H, Ubl JJ, Reiser G. 2002. Four subtypes of protease- activated receptor 2-mediated mitogenic pathway. J Biol Chem activated receptors, co-expressed in rat astrocytes, evoke different 272:7519–7524. physiological signaling. Glia 37:53–63.