Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can be cleaved by thrombin. As yet, however, little is known from studies of the receptor protein itself. To obtain such information, we have prepared monoclonal antibodies against a peptide corresponding to receptor residues Ser42 through Phe55, the domain immediately distal to the site of cleavage by thrombin. By flow cytometry, all of the antibodies reacted with the thrombin-responsive megakaryoblastic CHRF-288 and HEL cell lines, but not with the T-lymphoid Sup-T1 cell line. Functionally, the antibodies inhibited platelet responses to alpha-thrombin, gamma-thrombin, and trypsin, but had no effect on platelet activation by ADP, epinephrine, or the thromboxane analog U46619. Radioiodinated antibody bound to approximately 1,800 sites/platelet, a value similar to the reported number of moderate affinity thrombin binding sites per platelet. On Western blots, the antibodies recognized a 66-kDa protein in platelet, HEL, and CHRF-288 membranes. The discrepancy between this apparent size and the predicted mass of the receptor suggests that, as with other G protein-coupled receptors, one or more of the potential sites for N-linked glycosylation have been utilized. Therefore, these results suggest that: 1) the cloned thrombin receptor is involved in a broad range of platelet responses to thrombin, as well as gamma-thrombin and trypsin; 2) as predicted, the N terminus of the receptor is accessible on the platelet surface; 3) the moderate affinity thrombin binding site noted in earlier studies may be the receptor; 4) potentially as much as one third of the mass of the receptor is carbohydrate.
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PMID:Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. 132 Nov 25

Structure-activity studies on a series of analogues of N-(3-methyl-S-(1-pyrrolidinyl carbonyl) butyl)-D-alanine ethyl ester hydrochloride (SC42619) have defined the features of this dipeptide analogue required for observation of thrombin receptor antagonist activity on the human platelet. The affinity for SC42619, and for its structural analogue SC43583 is enhanced by pretreatment of the platelets with chymotrypsin. Endothelial cell prostacyclin (PGI2) synthesis induced by thrombin and trypsin is selectively inhibited by SC42619 provided that prolonged exposure to this antagonist is avoided. However inhibition of PGI2 synthesis by SC42619 is not overcome by increasing the thrombin concentration. The data provide further support for identification of SC42619 and certain of its analogues as selective antagonists at the platelet thrombin receptor but suggest that these compounds may have more complex, and possibly non-selective effects on the endothelial cell.
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PMID:Thrombin receptor antagonists. Structure-activity relationships for the platelet thrombin receptor and effects on prostacyclin synthesis by human umbilical vein endothelial cells. 215 30

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.
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PMID:Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells. 298 Dec 34

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.
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PMID:Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells. 613 25

We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.
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PMID:Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. 755 75

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.
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PMID:Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. 756 91

The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.
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PMID:Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. 771 46

The present study was undertaken to define clearly the receptor, which is responsible for the thrombin induced vWf release from HUVEC. Vu et al. reported that cleavage of the platelet thrombin receptor by thrombin resulted in a new N-terminus (SFLLRN...) which acts as tethered ligand (4). The free peptide activates platelets and induces rises in both cytosolic free Ca2+ and PGI2 production in HUVEC (10). HUVEC were incubated with thrombin, SFLLRN or other relevant substances. After incubation, the intracellular vWf content was compared with the vWf concentration in the supernatant. The intracellular vWf concentration was measured by microscope fluorometry and the concentrations in the supernatant with an ELISA. The thrombin stimulated cells showed 53% vWf antigen compared with control cells (100%). This result was well correlated with the 2.4 fold higher vWf concentrations measured in the supernatant of thrombin stimulated cells than in control cells. Also the addition of SFLLRN (1-60 microM) or trypsin (1-50 nM) increased vWf release from HUVEC in a dose dependent manner. These results indicate that thrombin induced vWf release from HUVEC is mediated through the activation of the tethered ligand receptor.
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PMID:The tethered ligand receptor is the responsible receptor for the thrombin induced release of von Willebrand factor from endothelial cells (HUVEC). 774 May 17

Cultures of vascular smooth muscle cells (VSMC) are commonly used to study the events and defects found in hypertension and atherosclerosis. In particular Ca2+ homeostasis in cellular signalling has been the focus of extensive research. Since trypsin has been shown to mobilise Ca2+ in some cell types, we have investigated its effect on various aspects of Ca2+ homeostasis in rat aortic smooth muscle cells (RASMC). The effects of trypsin, alpha-chymotrypsin and elastase (other serine proteases) on intracellular Ca2+ in cultured aortic cells isolated from Wistar rats have been investigated. Trypsin (24 micrograms/ml) elicits intracellular Ca2+ mobilisation, after which cells become nonresponsive to thrombin Ca2+ mobilisation but retain responsiveness to Angiotensin II (AII). alpha-Chymotrypsin (24 micrograms/m) inhibits the thrombin Ca2+ mobilising response, without itself initiating a Ca2+ transient or affecting AII Ca2+ mobilisation. Elastase (24 micrograms/ml) was not effective in mobilising intracellular Ca2+ or inhibiting the thrombin response. We have also observed diminished thrombin Ca2+ mobilisation responses between cells in suspension and cell monolayers, which appeared to be unrelated to proteolysis but due to morphological changes of the cells. Our results suggest that trypsin acts on the thrombin receptor via a specific proteolysis mechanism to mobilise intracellular Ca2+ ([Ca2+]i) in RASMC. The amount of Ca2+ released by thrombin or trypsin is dependent on the morphology of the cell and the state of the tethered ligand of the thrombin receptor exposed by the protease.
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PMID:The effect of thrombin and serine proteases on intracellular Ca2+ in rat aortic smooth muscle cells. 779 84

We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5' end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a separate exon located about 10 kilobases away from the 3' exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 microM PAR-2-activating peptide (SLIGRL), or 1 microM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.
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PMID:The mouse proteinase-activated receptor-2 cDNA and gene. Molecular cloning and functional expression. 789 Jul 26


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