Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors that play critical roles in thrombosis, inflammation, and vascular biology. PAR1 is proposed to be involved in the invasive and metastatic processes of various cancers. However, the protease responsible for activating the proinvasive functions of PAR1 remains to be identified. Here, we show that expression of PAR1 is both required and sufficient to promote growth and invasion of breast carcinoma cells in a xenograft model. Further, we show that the matrix metalloprotease, MMP-1, functions as a protease agonist of PAR1 cleaving the receptor at the proper site to generate PAR1-dependent Ca2+ signals and migration. MMP-1 activity is derived from fibroblasts and is absent from the breast cancer cells. These results demonstrate that MMP-1 in the stromal-tumor microenvironment can alter the behavior of cancer cells through PAR1 to promote cell migration and invasion.

Classical ligands bind to the extracellular surface of their cognate receptors and activate signaling pathways without crossing the plasma membrane barrier. We selectively targeted the intracellular receptor-G protein interface by using cell-penetrating membranetethered peptides. Attachment of a palmitate group to peptides derived from the third intracellular loop of protease-activated receptors-1 and -2 and melanocortin-4 receptors yields agonists and/or antagonists of receptor-G protein signaling. These lipidated peptides-which we have termed pepducins-require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor third intracellular loop but instead require the C-tail of the receptor. Construction of such peptide-lipid conjugates constitutes a new molecular strategy for the development of therapeutics targeted to the receptor-effector interface. G protein-coupled receptors (GPCRs) play a vital role in the signaling processes that control cellular metabolism, cell growth, and motility, inflammation, neuronal signaling, and blood coagulation. Although remarkably diverse in sequence and function, all GPCRs share a highly conserved topological arrangement of a seven-transmembrane helical core domain joined by three intracellular loops, three extracellular loops, and N-and C-terminal domains (1). A key event for the switch from inactive to active receptor is ligand-induced conformational changes of transmembrane helices 3 (TM3) and 6 (TM6) (2). These helical movements in turn alter the conformation of the intracellular loops of the receptor to promote activation of associated heterotrimeric G proteins.Mutagenesis studies (3-5) demonstrated that the third intracellular loop (i3) mediates a large part of the coupling between receptor and G protein. i3 loops expressed as minigenes have also been shown to directly compete with ␣1B-adrenergic receptors for G q binding (6). Okamoto and colleagues (7) localized a G protein activator region in the C-terminal end of the third cytoplasmic loop of the human ␤2-adrenergic receptor. They showed that a soluble peptide corresponding to this region (R 259 -K 273 ) activates G s protein under cell-free conditions. Moreover, related peptides found in wasp venom, such as mastoparan, stimulate GDP-GTP exchange from purified G proteins (8). These amphiphilic cationic peptides act in the absence of receptors to directly stimulate G i and G o and compete with intact receptor for the G protein ␣ subunit (9). However, there are currently no effective strategies to directly study the mechanism of receptor-G protein coupling in a controlled fashion under in vivo conditions.Here, we present an approach to study receptor-mediated G protein activation by using palmitoylated peptides as receptormodulating agents based on the i3 loops of the protease-activated receptors (PAR), PAR...

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.Sepsis remains the leading cause of mortality of patients in intensive care units, causing at least 210,000 deaths annually in the United States1. Much of the pathology of sepsis has been attributed to a hyper-reaction of the inflammatory system to the invading pathogens, a condition called 'systemic inflammatory response syndrome' 2 . During the early phases of sepsis, systemic concentrations of inflammatory cytokines and chemokines rapidly increase and the endothelium is activated to cause vascular leakage and septic shock. In late-stage sepsis, the clotting cascade is triggered by the damaged endothelium, leading to disseminated intravascular coagulation (DIC) and multiorgan failure 3, 4. The vascular damage is caused by many sepsis-related factors, including bacterial endotoxin, tumor

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.

Abstract-Thrombosis associated with the pathophysiological activation of platelets and vascular cells has brought thrombin and its receptors to the forefront of cardiovascular medicine. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiological responses including platelet activation, intimal hyperplasia, inflammation, and maintenance of vascular tone and barrier function. The thrombin receptors PAR1 and PAR4 can be effectively targeted in animals in which acute or prolonged exposure to thrombin leads to thrombosis and/or restenosis. In the present study, we describe the molecular and pharmacological basis of small-molecule inhibitors that target PAR1. In addition, we discuss a new class of cell-penetrating inhibitors, termed pepducins, that provide insight into previously unidentified roles of PAR1 and PAR4 in protease signaling. Key Words: arteries Ⅲ endothelium Ⅲ inhibitors Ⅲ platelets Ⅲ receptors Ⅲ signal transduction Ⅲ thrombosis P rotease-activated receptors (PARs) play critical roles in coagulation, inflammation, and vascular homeostasis. [1][2][3][4][5] Proteases that are produced during vascular injury exert many of their cellular effects by cleaving and activating the PARs. Thrombin-dependent platelet activation and aggregation have been shown to be heightened in the setting of angioplasty and stenting, which may cause clinical complications including acute myocardial infarction and death. 6 -8 The high-affinity thrombin receptor PAR1 has long been recognized as an obvious candidate for therapeutic intervention in patients with acute coronary syndromes. It is not yet known, however, whether targeting only PAR1 will achieve sufficient therapeutic efficacy because of the presence of a more recently identified second thrombin receptor named PAR4. 9 -12 PAR1 and PAR2 (a trypsin but not a thrombin receptor) have also been shown to affect other cardiovascular functions such as vasoreactivity and cardiomyocyte hypertrophy.The purpose of the present review is to help the clinical reader understand why PARs are essential for the maintenance of normal vascular integrity. This review will focus on the potential therapeutic utility of targeting the PARs in thrombosis, atherosclerosis, and restenosis. Historically, the PARs have been recalcitrant to the development of peptidomimetic-based antagonists; however, recent PAR1 drug candidates based on natural products are now entering large-scale clinical trials for treatment of patients with acute coronary syndromes. In an orthogonal approach, PARs have also been blocked on the inside of the cell with the use of cell-penetrating pepducins that prevent signaling to internally located G proteins. [13][14][15][16][17] Proof-of-concept experiments in acute thrombosis models point to novel antiplatelet therapies that could potentially benefit patients at risk for acute thrombosis. The Role of PARs in Normal Platelet FunctionPlatelets are essential for proper blood coagulation. Initiation of a platelet thromb...

Background-Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. Methods and Results-Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. Conclusions-We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.