Showing papers on "Protease-activated receptor 2 published in 1999"

How the protease thrombin talks to cells.

Abstract: How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1's proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naive receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways.

Abstract: The traditional view of growth factor receptors and hormone receptors in general is that a specific ligand directly recognizes a highly selective binding site on its cognate receptor and, thereby, activates receptor-dependent signaling and biological responses. In the case of the EGF receptor, several structurally related proteins (EGF, transforming growth factor alpha, amphiregulin, betacellulin, epiregulin, heparin-binding EGF) are recognized as direct agonists. Each of these growth factors binds to the ectodomain of the EGF receptor and provokes its activation through a mechanism that involves dimerization, activation of the receptor tyrosine kinase cytosolic domain, and autophosphorylation of the receptor. This process initiates signaling pathways that lead to mitogenesis. Recently it has become apparent that the EGF receptor is also part of signaling networks activated by stimuli that do not directly interact with this receptor. These stimuli include agonists that specifically bind to other membrane receptors, membrane depolarization agents, and environmental stressors. The data not only show EGF-independent tyrosine phosphorylation of the EGF receptor, but also provide experimental evidence that the EGF receptor participates in the signaling events and cellular responses initiated by these various stimuli. Collectively, the results imply that in the absence of direct agonists the EGF receptor is employed in a wide array of biological signaling processes. The purpose of this article is to review and evaluate these nonclassical uses of the EGF receptor.

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

Abstract: The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identificiation of its cDNA.

The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2.

Abstract: Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1.

Abstract: We developed mice deficient in protease-activated receptor-2 (PAR-2) or PAR-1 to explore the pathophysiological functions of these receptors. In this report, we evaluated mean arterial pressure and heart rate (HR) changes in response to PAR-1 or PAR-2 activation in anesthetized wild-type (WT), PAR-1-deficient (PAR-1-/-), and PAR-2-deficient (PAR-2-/-) mice. In WT mice, TFLLRNPNDK, a PAR-1 selective activating peptide, caused hypotension and HR decreases at 1 μmol/kg. TFLLRNPNDK also caused secondary hypertension followingl-NAME pretreatment. These responses were absent in PAR-1−/− mice. In WT mice, SLIGRL, a PAR-2 selective activating peptide, caused hypotension without changing HR at 0.3 μmol/kg. SLIGRL did not induce hypertension following N ω-nitrol-arginine-methyl ester-HCl (l-NAME). The response to SLIGRL was absent in PAR-2-/- mice. SFLLRN, a nonselective receptor activating peptide caused hypotension and HR decreases in WT mice at 0.3 μmol/kg, as well as secondary hypertension followingl-NAME. SFLLRN still induced hypotension in PAR-1−/− mice, but HR decrease and secondary hypertension following l-NAME were absent. The hypotensive and bradycardic responses to SFLLRN and TFLLRNPNDK in PAR-2−/− mice were accentuated compared with WT mice. By using mouse strains deficient in either PAR-1 or PAR-2, we confirmed the in vivo specificity of TFLLRNPNDK and SLIGRL as respective activating peptides for PAR-1 and PAR-2, and the distinct hemodynamic responses mediated by activation of PAR-1 or PAR-2. Moreover, the accentuated response to PAR-1 activation in PAR-2-deficient mice suggests a compensatory response and potential receptor cross-talk.

Intracellular sigma1 receptor modulates phospholipase C and protein kinase C activities in the brainstem.

Abstract: Most physiological effects of σ1 receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the σ1 receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the σ1 receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C β1 and β2 isoforms and the phosphorylation of a protein of the same molecular weight as the cloned σ1 receptor lead to a desensitization of the σ1 motor response. Our results indicate that the intracellular σ1 receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses.

Protease-Activated Receptor-2 Involvement in Hypotension in Normal and Endotoxemic Rats In Vivo

Abstract: Background—The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. Methods and Results—Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin–SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PA...

Novel Actions of Inverse Agonists on 5-HT2C Receptor Systems

Abstract: In cell systems where ligand-independent receptor activity is optimized (such as when receptors are overexpressed or mutated), acute treatment with inverse agonists reduces basal effector activity whereas prolonged exposure leads to sensitization of receptor systems and receptor up-regulation. Few studies, however, have reported effects of inverse agonists in systems where nonmutated receptors are expressed at relatively low density. Here, we investigated the effects of inverse agonists at human serotonin (5-HT)2C receptors expressed stably in Chinese hamster ovary cells (≈250 fmol/mg protein). In these cells, there is no receptor reserve for 5-HT and 5-HT2C inverse agonists did not reduce basal inositol phosphate (IP) accumulation nor arachidonic acid (AA) release but behaved as simple competitive antagonists, suggesting that these receptors are not overexpressed. Prolonged treatment (24 h) with inverse agonists enhanced selectively 5-HT2C-mediated IP accumulation but not AA release. The enhancing effect occurred within 4 h of treatment, reversed within 3 to 4 h (after 24-h treatment), and could be blocked with neutral antagonists or weak positive agonists. The enhanced responsiveness was not due to receptor up-regulation but may involve changes in the expression of the G protein, Gαq/11 and possibly Gα12 and Gα13. Interestingly, 24-h exposure to inverse agonists acting at 5-HT2C receptors also selectively enhanced IP accumulation, but not AA release, elicited by activation of endogenous purinergic receptors. These data suggest that actions of inverse agonists may be mediated through effects on receptor systems that are not direct targets for these drugs.

Methods of using human receptor protein 4-1bb

Abstract: Disclosed herein are methods of using the H4-1BB protein, ligands to this protein, and various mAbs either directed against H4-1BB or other molecules that can be used therapeutically. The nature and importance of the H4-1BB molecule provides the ligands and related co-stimulatory molecules the ability to enhance or suppress T cell activation and proliferation. By treating T cells that have expressed receptor protein H4-1BB with one of the four anti-H4-1BB monoclonal antibodies disclosed herein, activation or inhibition of the immune response is seen. Also disclosed herein is cDNA for the human receptor H4-1BB. The cDNA of the human receptor H4-1BB is about 65 % homologous to the mouse cDNA 4-1BB and was isolated by using probes derived from murine cDNA 4-1BB. A fusion protein for detecting cell membrane ligands to human receptor protein H4-1BB was developed. It comprises the extracellular portion of the receptor protein H4-1BB and a detection protein, alkaline phosphatase, bound to the portion of the receptor protein H4-1BB. B cells that have expressed a ligand to receptor protein H4-1BB can be treated with cells that have expressed receptor protein H4-1BB and B cell proliferation may be induced. The use of H4-1BB to block H4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation or against autoimmune diseases including diabetes, rheumatoid arthritis, and lupus. Other applications of this technology include the development of therapeutic methods for the treatment of HIV-1 infected individuals, and the treatment of cancerous tumors.

Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Abstract: Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2 Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm–egg binding Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed

Discovery of novel non-peptide CCR1 receptor antagonists.

Abstract: Ligands for the CCR1 receptor (MIP-1alpha and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1alpha and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Angiotensin II type 1 and type 2 receptors bind angiotensin II through different types of epitope recognition.

Abstract: ObjectiveThis study was designed to demonstrate that the principle of molecular recognition underlying high-affinity binding of angiotensin II to the type 2 (AT2) receptor is distinct from that of the type 1 (AT1) receptor. In general, the same functional pharmacophores in hormones are used to bind

Evidence for Selective Effects of Chronic Hypertension on Cerebral Artery Vasodilatation to Protease-Activated Receptor-2 Activation

Abstract: Background and Purpose—Protease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH2). PAR-2 mediates nitric oxide (NO)–dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2–mediated vasodilatation is similarly adversely affected by this disease state. Methods—We studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY v...

Regulation of D 1 Dopamine Receptors with Mutations of Protein Kinase Phosphorylation Sites: Attenuation of the Rate of Agonist-Induced Desensitization

Abstract: Investigations of D1 receptor regulation have suggested a role for cAMP-dependent protein kinase (PKA) in agonist-induced desensitization and down-regulation of receptor expression. Given the presence of at least four possible consensus recognition sites for PKA on the D1 receptor protein, a reasonable hypothesis is that some of these PKA-mediated effects are caused by phosphorylation of the receptor. As an initial test of this hypothesis, we used site-directed mutagenesis to create a mutant D1 receptor with substitutions at each of its four potential PKA phosphorylation sites. The modified amino acids are as follows: Thr135 to Val, Ser229 to Ala, Thr268 to Val, and Ser380 to Ala. Characterization of the wild-type and mutant receptors stably expressed in C6 glioma cells suggests that the mutations have no effect on receptor expression, antagonist or agonist affinities, or on functional coupling with respect to cAMP generation. Similarly, dopamine preincubation of the stably transfected C6 cells expressing either the wild-type or mutated D1 receptors results in an agonist-induced loss of ligand binding activity (down-regulation) in an identical fashion. In contrast, the time of onset of dopamine-induced desensitization is greatly attenuated in the quadruple mutant receptor. After 1 h of dopamine pretreatment, the wild-type receptor exhibits ∼80% desensitization of the cAMP response, whereas the mutant receptor is desensitized by only ∼20%. Further analyses of single mutated receptors, in which only one of the four putative phosphorylation sites is modified, reveals that Thr268 in the third cytoplasmic loop of the receptor protein is primarily responsible for regulating the desensitization kinetics. These results are consistent with the hypothesis that phosphorylation of the D1 receptor on Thr268 is important for rapid agonist-induced homologous desensitization.

Increased expression of protease activated receptor-2 (PAR-2) in balloon-injured rat carotid artery.

Abstract: Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohistochemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.