Showing papers on "Protease-activated receptor 2 published in 1993"

Interleukin 1 signaling occurs exclusively via the type I receptor.

Abstract: Two receptors for the proinflammatory cytokine interleukin 1 (IL-1) have been cloned and characterized biochemically. While it has been well established that the type I (80-kDa) IL-1 receptor can mediate responses to IL-1, the function of the type II (60-kDa) IL-1 receptor has been unknown. In this manuscript we describe experiments designed to ask whether the type II receptor is capable of delivering a biological signal. We have examined two types of experimental situation: responses to IL-1 in cells which express predominantly the type II receptor, and responses to IL-1 which have been suggested previously in the literature to be mediated by type II receptors. In both situations we find that the responses instead are mediated via type I receptors. A blocking antibody against the type II receptor never inhibits, and in fact sometimes enhances, the responses. We conclude that a very small number of type I receptors is sufficient to mediate all of the actions of IL-1 which we have examined here and that the function of the type II receptor may not be to transduce signals.

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.

Abstract: A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatics islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity ( K d = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9–39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9–39) was an antagonist of the receptor, inhibiting GLP-1–induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.

Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase.

Abstract: Serotonin modulates a wide range of physiological functions by activating multiple receptors, which are coupled to various effector systems. Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors, we have isolated from a mouse brain library a cDNA encoding a new 5-HT receptor, 5-HTx, that activates adenylate cyclase. Amino acid sequence comparisons revealed that the 5-HTx receptor was a distant relative of previously cloned 5-HT receptors, with the highest percentage of homology (42%) being with the 5-HTdro1 receptor, a Drosophila 5-HT receptor positively coupled to adenylate cyclase. In COS-7 cells transiently expressing the 5-HTx receptor, 5-HT induced an increase in cAMP levels that was dose dependent and saturable (EC50 = 45 nM). Agonists displayed the following rank order of potencies: 5-carboxamidotryptamine > 5-methoxytryptamine > 5-HT > RU 24969 > 8-hydroxy-2-(di-n-propylamino)tetralin. The most efficient antagonists in inhibiting the stimulatory effect of 5-HT were methysergide, methiothepin, mesulergine, metergoline, clozapine, ergotamine, and (+)-butaclamol. Membranes of COS-7 cells expressing the 5-HTx receptor displayed a single saturable binding site for [3H]5-HT. The order of potencies of various drugs in displacing [3H]5-HT binding was similar to the order obtained in cAMP experiments. The pharmacological profile of this receptor does not correspond to the profile of any of the classic 5-HT receptor subtypes. Expression of 5-HTx mRNA was highest in brainstem and lower in forebrain, cerebellum, intestine, and heart. The 5-HTx receptor might therefore correspond to 5-HT1-like receptors that have been shown to induce relaxation in porcine vena cava and guinea pig ileum as well as tachycardia in cat heart. The high affinity of the 5-HTx receptor for neuroleptic agents such as (+)-butaclamol and clozapine suggests also that this receptor might play a role in certain neuropsychiatric disorders.

Cross-coupling of signal transduction pathways: the dioxin receptor mediates induction of cytochrome P-450IA1 expression via a protein kinase C-dependent mechanism.

Abstract: Signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug-metabolizing enzymes, such as cytochrome P-450IA1 and glutathione S-transferase Ya. Exposure of the dioxin receptor to dioxin leads to an apparent translocation of the receptor to the nucleus in vivo and to a rapid conversion of the receptor from a latent, non-DNA-binding form to a species that binds to dioxin-responsive positive control elements in vitro. This DNA-binding form of receptor appears to be a heterodimeric complex with the helix-loop-helix factor Arnt. In this study, we show that activation of the cytochrome P-450IA1 gene and minimal dioxin-responsive reporter constructs by the dioxin receptor was inhibited following prolonged treatment of human keratinocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Inhibition of the receptor-mediated activation response was also achieved by treatment of the cells with a number of protein kinase inhibitors, one of which, calphostin C, shows selectivity for protein kinase C. Taken together, these data suggest that protein kinase C-dependent phosphorylation may play an essential role in the dioxin signaling pathway. This hypothesis is supported by the observation that pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate inhibited the DNA-binding activity of the dioxin receptor in vivo. In vivo, the dioxin receptor was found to be a phosphoprotein. In vitro, dephosphorylation of the ligand-activated, heteromeric dioxin receptor form or dephosphorylation of the individual ligand-binding and Arnt receptor subunits inhibited the xenobiotic response element-binding activity. Moreover, dephosphorylation experiments with the individual receptor subunits prior to assembly of the xenobiotic response element-binding receptor form indicated that phosphorylation seemed to be important for the DNA-binding activity per se of the receptor, whereas Arnt appeared to require phosphorylation to interact with the receptor. Finally, a protein kinase C inhibitor-sensitive cytosolic catalytic activity that could restore the DNA-binding activity of the dephosphorylated dioxin receptor form was identified.

Antibodies to human IL-8 type A receptor

Abstract: cDNAs encoding a class of receptors, including the IL-8 type B receptor, have been identified in human tissue. Recombinantly produced IL-8 type B receptor is used in the preparation and purification of antibodies capable of binding to the receptor, and in diagnostic assays. The antibodies are advantageously used in the prevention and treatment of inflammatory conditions.

Serotonin receptor cDNA cloned from Lymnaea stagnalis.

Abstract: Serotonin (5-HT) is a major neurotransmitter that influences various behaviors, neuronal plasticity, learning, and memory in molluscs. Although the physiology of 5-HT transmission in molluscs is well studied, little is known about the pharmacology and diversity of the 5-HT receptor system. Based on the high homology of genes coding for guanine nucleotide-binding protein (G protein)-coupled receptors, we have cloned a gene for the Lymnaea stagnalis 5-HT (5HTlym) receptor. The putative receptor protein, 509 amino acids long, has highest homology with the Drosophila 5-HT receptors and mammalian 5HT1 receptors. As revealed by RNA blot-hybridization analysis, two mRNA species of 2.3 and 3.2 kb are detected in the central nervous system of Lymnaea. Transient expression of 5HTlym in COS-7 cells showed saturable [3H]lysergic acid diethylamide binding with an estimated dissociation constant of 0.9 nM. The 5HTlym receptor exhibited a mixed 5HT-like pharmacology that cannot be precisely categorized with existing mammalian classification nomenclature. However, the 5HTlym receptor does display some characteristics that have been attributed to the putative mammalian vascular 5HT1-like receptor.

Characterization of the human beta 3-adrenergic receptor gene.

Abstract: Comparison of the rodent and human beta 3-adrenergic receptor cDNAs with the respective genomic sequences has revealed unexpectedly that these genes contain two protein-coding exons. The rat gene was cloned recently and was found to contain three exons and two introns. In the present report, the human beta 3 receptor gene was characterized and was found to consist of two exons and a single intron. Sequence analysis of the human beta 3 receptor gene identified regions in the intron that were homologous to the second exon and second intron of the rat gene. It appears that both species utilize homologous 5' donor sites in the first intron and 3' acceptor sites of the final exon. However, splicing signals within the human intron that are homologous to the second exon of the rat gene are not used. Nuclease protection assays of tissue RNA and polymerase chain reaction-amplified cDNA demonstrated conclusively that beta 3 receptor mRNA, containing two protein-coding exons, is expressed in human adipose and intestinal tissues. The pharmacological properties of the full length human beta 3 receptor were determined for the first time in Chinese hamster ovary cells, where catecholamine agonists activated adenylyl cyclase with low potency. The beta 3 receptor agonists CGP 12177 and BRL 37344 also activated adenylyl cyclase. CGP 12177 was 10-15 times more potent than either isoproterenol or BRL 37344 in stimulating adenylyl cyclase activity. These pharmacological properties differed somewhat from those reported previously for Chinese hamster ovary cells expressing the truncated receptor. However, direct comparison indicates that it is unlikely that the amino acid sequence derived from the second exon can account for these differences.

Activation of oligomerizing receptors by using fused receptor ligands

Abstract: The invention concerns a method for activating receptors selected from receptor tyrosine kinases, cytokine receptors and members of the nerve growth factor receptor superfamily. A conjugate comprising the direct fusion of at least two ligands capable of binding to the receptor(s) to be activated is contacted with the receptors, whereby the ligands bind their respective receptors inducing receptor oligomerization.

Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-γ

Abstract: The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.

Response of blood leukocytes to thrombin receptor peptides.

Abstract: Thrombin has receptor-mediated effects on a variety of cell types. A recently cloned platelet thrombin receptor exerts its effects by a tethered-ligand mechanism. A similar receptor was shown in at least two nonplatelet cell types, fibroblasts and endothelial cells. Thrombin has biologically important effects on leukocytes, but the type of receptor mediating the effects is not known. Therefore, we examined the responses of monocytes, neutrophils, and lymphocytes to thrombin and to an agonist specific for the platelet-type thrombin receptor. We compared the effects of a peptide (SFLLRNPNDKYEPF) corresponding to residues 42-55 of the cloned platelet thrombin receptor on calcium flux in platelets and leukocytes. The thrombin receptor peptide induced increases in intracellular calcium in platelets and monocytes that reached a maximum at 5 microM peptide. The maximal increase was similar in magnitude to the response to thrombin. Lymphocytes showed a small and variable increase in intracellular calcium in response to thrombin or the thrombin receptor agonist. The thrombin receptor peptide had no effect on neutrophil calcium concentrations. When the amino acid corresponding to Arg 46 was replaced with Ala in the synthetic peptide, the ability to increase intracellular calcium was abolished for both platelets and monocytes. The peptide instead had thrombin antagonist activity. Thus, monocytes respond to thrombin receptor peptides similarly to platelets. We conclude that human monocytes possess a thrombin receptor similar to that present on platelets. Furthermore, the residue corresponding to Arg 46 of the thrombin receptor is critical for receptor agonist activity.

Isolation and expression of a novel angiotensin II receptor from Xenopus laevis heart.

Abstract: A Xenopus laevis heart cDNA library was screened using the human angiotensin type 1 (AT1) receptor cDNA coding sequence as a hybridization probe. A cDNA was isolated that encodes a protein of 363 amino acids that shares 63% sequence identity with the human AT1 receptor. Radioligand binding studies with the cloned receptor expressed in COS cells indicated that it is an angiotensin II receptor that possesses pharmacological properties distinct from those of the two known mammalian receptor subtypes, AT1 and AT2. Electrophysiological studies with the recombinant receptor expressed in X. laevis oocytes revealed that the amphibian receptor, like the mammalian AT1 receptor, can functionally couple to a second messenger system, leading to the mobilization of intracellular stores of calcium. However, nonpeptide antagonists selective for the mammalian AT1 and AT2 receptors do not block angiotensin II-stimulated functional responses in injected oocytes, which confirms that the amphibian receptor is a pharmacologically unique angiotensin II receptor. Nevertheless, based on conservation of structural features and motifs and similarity in coupling mechanisms, we speculate that the cloned Xenopus receptor is the amphibian counterpart of the mammalian AT1 receptor, having acquired its unique pharmacology as a consequence of evolutionary divergence.

Production of the serum form of the transferrin receptor by a cell membrane-associated serine protease.

Abstract: Recent investigations have demonstrated that the soluble form of the transferrin receptor in human serum is an 85-kDa fragment of intact receptor containing most of the extracellular domain. The recent demonstration of a remnant of the truncated receptor in cell membranes suggests that the soluble form arises from proteolytic cleavage of intact receptor. In the present investigation, domain-specific antibodies were used to further examine the subcellular location and nature of this proteolysis. HL60 cells were used in the investigation because the cells release an 85-kDa soluble form of the receptor to the culture supernatant that is identical to that found in serum. When intact, purified transferrin receptor from human placenta was incubated with the culture supernatant, no proteolytic activity could be demonstrated. However, when purified membrane fractions from HL60 cells were used in this incubation system, the 85-kDa fragment was produced. This activity was inhibited by serine protease inhibitors indicating that cell membrane fractions contain a serine protease capable of producing the serum form of the transferrin receptor.

Mammalian receptors for interleukin-10 (il-10)

Abstract: Mammalian IL-10 receptor subunits are provided, together with nucleic acids encoding various species variants of the subunits. Uses of the nucleic acids and receptor subunits are also provided, including methods for screening for agonists and antagonists of the receptor ligands, methods for producing diagnostic or therapeutic reagents, and methods for producing antibodies. Therapeutic or diagnostic reagents and kits are also provided.

Non-catalytic activation of phospholipase C-gamma 1 in vitro by epidermal growth factor receptor.

Abstract: To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.

The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling.

Abstract: It has previously been reported that murine macrophages can respond chemotactically and mitogenically to the serine proteinase thrombin There is a similar response in these macrophages to catalytically inactivated thrombin or to peptide fragments of the thrombin B-chain [Bar-Shavit, Kahn, Mann and Wilner (1986) Proc Natl Acad Sci USA 83, 976-980] However, the existence of a non-proteolytic mechanism of thrombin receptor activation in mononuclear cells was not evident in the present study using U937 human monocytic cells The ability of thrombin to stimulate intracellular Ca2+ mobilization, actin polymerization or cell proliferation was not mimicked by N alpha-tosyl-L-lysine chloromethyl ketone (TLCK)-treated thrombin or by a synthetic 14-amino-acid peptide (single amino acid letter code YPPWNKNFTENDLL) corresponding to a part of the B-chain of thrombin which was reported to be mitogenic in murine macrophages Evidence was obtained, however, in U937 cells for the presence of proteolytic-dependent thrombin receptor similar to the thrombin receptor expressed in platelets, which following thrombin cleavage exposes a new N-terminal tethered ligand In support of this, a thrombin-receptor-derived hexapeptide (TRP; sequence SFLLRN), corresponding to a part of the thrombin receptor tethered ligand, mimicked all the actions of thrombin in U937 cells Further, TRP and thrombin cross-desensitized U937 cells to subsequent stimulation with either TRP or thrombin, suggesting that TRP acted through the same U937 cell surface receptor as did thrombin Thrombin activation of U937 monocytic cells can therefore be accounted for entirely by a proteolytic mechanism of thrombin receptor activation