Showing papers on "Protease-activated receptor 2 published in 1996"

The Cytolytic P2Z Receptor for Extracellular ATP Identified as a P2X Receptor (P2X7)

Abstract: The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

Role of the thrombin receptor in development and evidence for a second receptor

Abstract: Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, endothelial cells, leukocytes and mesenchymal cells. A G-protein-coupled receptor that is proteolytically activated by thrombin is a target for drug development aimed at blocking thrombosis, inflammation and proliferation. Here we show that although disruption of the thrombin receptor (tr) gene in mice causes about half of the tr-/- embryos to die at embryonic day 9-10, half survive to become grossly normal adult mice with no bleeding diathesis. Strikingly, tr-/- platelets respond strongly to thrombin, whereas tr-/- fibroblasts lose their ability to respond to thrombin. We conclude that the thrombin receptor plays an unexpected role in embryonic development, suggesting a possible new function for the 'coagulation' proteases themselves. Moreover, a second platelet thrombin receptor exists, and different thrombin receptors have tissue-specific roles. This may allow development of therapeutics that will selectively block thrombin's different cellular actions.

Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor.

Abstract: Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).

Identification of competitive antagonists of the P2Y1 receptor.

Abstract: Although P2 receptors mediate a myriad of physiological effects of extracellular adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist molecules. The adenine nucleotide-promoted inositol lipid hydrolysis response of turkey erythrocyte membranes, which has been used extensively as a model for P2Y receptors, has been applied to identify molecules that competitively block these receptors. Adenosine-3'-phosphate-5' -phosphosulfate (A3P5PS) promoted activation of phospholipase C that was only 10-25% of that observed with the full P2Y receptor agonists ATP, ADP, and 2-methylthio-ATP (2MeSATP). The small stimulatory effects of A3P5PS were saturable. Moreover, these effects were entirely the result of interaction with the P2Y receptor, because A3P5PS had no effect on activation of phospholipase C through the beta-adrenergic receptor and produced a concentration-dependent inhibition of 2MeSATP-promoted activity over the same range of A3P5PS concentrations that alone caused a small activation of phospholipase C. Increasing concentrations of A3P5PS produced a rightward shift of the concentration-effect curve for 2MeSATP, and Schild transformation of these data revealed that A3P5PS is a competitive P2Y receptor antagonist with a pKB of 6.46 +/- 0.17. The presence of a phosphate in the 2'- or 3'-position appears to be crucial for antagonist activity, because adenosine-3' -phosphate-5'- phosphate (A3P5P) and adenosine-2'- phosphate-5'-phosphate also exhibited competitive antagonist/partial agonist activities. Other 3'-substituted analogues, such as 3'-amino-ATP and 3'-benzoylbenzoyl-ATP, were full agonists with no antagonist activity. A3P5PS, A3P5P, and adenosine-2',5'-diphosphate also were competitive antagonists in studies with the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells. Moreover, both A3P5PS and A3P5P were devoid of agonist activity at the human P2Y1 receptor. The effects of these 2'- and 3'-phosphate analogues were specific for the phospholipase C-coupled P2Y1 receptor, because no agonistic or antagonistic effects on the adenylyl cyclase-coupled P2Y receptor of C6 glioma cells or on P2Y2, P2Y4, or P2Y6 receptors stably expressed in 1321N1 human astrocytoma cells were observed. These results describe specific competitive antagonism of the P2Y1 receptor by an adenine nucleotide derivative and provide a potential new avenue for P2 receptor drug development.

A lysine residue of the cannabinoid receptor is critical for receptor recognition by several agonists but not WIN55212-2.

Abstract: Lys192 in the third transmembrane domain of the human CB1 cannabinoid receptor was converted to an alanine to study its role in receptor recognition and activation by agonists. HU-210, CP-55940, WIN55212-2, and anandamide, four cannabinoid agonists with distinct chemical structures, were used to characterize the wild-type and the mutant receptors. In human embryonal kidney 293 cells stably expressing the wild-type receptor, specific binding to [3H]WIN55212-2 and inhibition of cAMP accumulation by cannabinoid agonists were demonstrated, with different ligands exhibiting the expected rank orders of potency and stereoselectivity in competition binding and functional assays. In cells expressing the mutant receptor, the binding affinity of the receptor for [3H]WIN55212-2 was only slightly affected (the Kd for the mutant receptor was twice that of the wild-type), and the ability of WIN55212-2 to inhibit cAMP accumulation was unchanged. However, HU-210, CP-55940, and anandamide were unable to compete for [3H]WIN55212-2 binding to the mutant receptor. In addition, the potencies of HU-210, CP-55940, and anandamide in inhibiting cAMP accumulation were reduced by > 100-fold. These results demonstrate that Lys192 is critical for receptor binding by HU-210, CP-55940, and anandamide. Because Lys192 is not important for receptor binding and activation by WIN55212-2, WIN55212-2 must interact with the cannabinoid receptor through at least one point of interaction that is distinct from those of the three other agonists.

Ubiquitination of the yeast a-factor receptor.

Abstract: The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

Abstract: We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

Abstract: We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Thrombin and trypsin-induced Ca(2+) mobilization in human T cell lines through interaction with different protease-activated receptors.

Abstract: This study was conducted to determine whether serine proteinases may induce [Ca(2+)]i mobilization in different hematopoietic cell lines and to analyze their mechanisms of action. We show that in addition to thrombin and thrombin receptor agonist peptide (TRP, SFLLRN), trypsin induced [Ca(2+)]i mobilization in a highly thrombin-sensitive Jurkat T cell clone. Thrombin, TRP, and trypsin were found to induce [Ca(2+)]i release in three different Jurkat T cell clones differing in the level of T cell receptor expression. Similar results were obtained with a prothymocytic leukemic cell line, HPB.ALL, although these cells were much more responsive to trypsin than to thrombin and TRP. Other cell types such as THP1, a myelomonocytic cell line, or CEM, a CD4(+) positive leukemic cell line, were unresponsive to thrombin, TRP, and trypsin. The effect of trypsin was mimicked by SLIGRL, a peptide corresponding to the cleaved amino-terminal sequence of the recently characterized murine trypsin-activated receptor (PAR2). At suboptimal concentrations, the effects of SFLLRN and SLIGRL were additive, whereas saturating doses of peptides did not further increase [Ca(2+)]i mobilization in Jurkat T cells, indicating that both peptides were able to mobilize the same pool of calcium. Northern blot analysis of mRNAs from different leukemic cell lines indicated a remarkable correlation between PAR2 expression in different cell lines and SLIGRL or trypsin responses in the same cells. The expression of the "trypsin receptor" was also confirmed by polymerase chain reaction analysis. Moreover, a 24 h treatment of Jurkat cells by an anti-CD3 monoclonal antibody, a condition known to down-regulate thrombin receptor expression, induced loss of thrombin and TRP responses but only partially affected trypsin stimulation of [Ca(2+)]i release. Finally, after a first stimulation with either thrombin or trypsin, Jurkat cells were still able to respond to trypsin or thrombin, respectively, demonstrating that thrombin and trypsin essentially activated their own receptors. Our data provided evidence that 1) the human T leukemic cell line Jurkat and other T cell lines express at least two different functional protease-activated receptors, the thrombin receptor and a highly sensitive trypsin receptor, likely the human counterpart of the murine PAR2, and 2) at variance with the commonly accepted model, trypsin exerts most of its effect in T leukemic cell lines by thrombin receptor-independent mechanisms.

A fully active nonglycosylated V2 vasopressin receptor

Abstract: The human V2 vasopressin receptor belongs to the superfamily of G protein-coupled receptors believed to be anchored to the plasma membrane by seven transmembrane regions. The extracellular portion of the human V2 vasopressin receptor contains one site susceptible to N-linked glycosylation. Metabolic labeling and immunoprecipitation of the receptor expressed in transfected cells were applied to examine whether the protein was indeed glycosylated. The V2 vasopressin receptor expressed transiently was glycosylated, but glycosidase treatment to test the complexity of the sugar moiety linked to asparagine revealed that the majority of the receptor protein lacked complex carbohydrates, an indication of an improperly processed protein. This immature protein displayed a tendency to form aggregates. In contrast with these data, testing of the sugar complexity of the receptor protein synthesized in stably transfected cells identified the predominant form as an appropriately processed receptor protein. Mutagenesis of asparagine 22 to glutamine produced on expression in transfected cells a nonglycosylated receptor with ligand binding affinity and coupling characteristics almost identical to those of the wild-type form. After exposure to elevated concentrations of AVP (100 nM), the nonglycosylated form desensitized to the same extent as the wild-type receptor.

Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

Abstract: In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.