Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.
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PMID:Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts. 1108 36

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 microM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 microM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.
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PMID:Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2. 1153 Dec 66

The human thromboxane A(2) (TP) receptor, a member of the G protein-coupled receptor superfamily, consists of seven transmembrane segments. Attempts to elucidate the specific segment(s) that define the receptor ligand-binding pocket have produced less than definitive and sometimes conflicting results. On this basis, the present work identified an amino acid sequence of the TP receptor that is directly involved in ligand binding. Mapping of this domain was confirmed by two separate approaches: photoaffinity labeling and site-specific antibodies. The newly synthesized, biotinylated photoaffinity probe, SQBAzide, was first shown to specifically label TP receptor protein. Sequential digestion of this protein with CNBr/trypsin revealed photolabeling of a 2.9-kDa peptide. Using anti-peptide antibodies directed against different regions of the receptor protein, it was established that this peptide represents the predicted cleavage product for CNBr/trypsin and corresponds to amino acids Arg(174)-Met(202) of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys(183)-Asp(193) was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly(172)-Cys(183) was not. Collectively these findings provide evidence that ligands interact with amino acids contained within the C-terminal portion of the third extracellular domain (ED3) of the receptor protein. This information should be of significant value in the study of TP receptor structure and signaling.
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PMID:Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. 1187 12

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.
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PMID:Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling. 1199 8

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.
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PMID:Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells. 1205 39

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.
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PMID:Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion. 1223 4

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.
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PMID:Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity. 1242 30

The proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. To clarify the presence of PAR-2 in human pancreatic cancer, the expression of PAR-2 was analyzed by RT-PCR, immunoblotting and immunocytochemistry using 5 human pancreatic cancer cell lines. And to evaluate the biological significance, immunohistochemical expression of PAR-2 in malignant and non-malignant human pancreatic tissues was examined using paraffin-embedded sections. The presence of PAR-2 was confirmed in all 5 pancreatic cancer cell lines and all 21 paraffin-embedded specimens from human pancreatic cancer examined. The expression of PAR-2 was found to be higher in the tissues with infiltrative growth pattern than those with expansive growth pattern. Moreover, significantly higher expression of PAR-2 was observed in the tissues which were accompanied by severe fibrosis. Even in the same specimen, the intensity of immunoreactivity tended to be stronger in the part with severe fibrosis than that with mild fibrosis. Similarly, the higher expression of PAR-2 was observed in chronic pancreatitis with severe fibrosis than with mild fibrosis. Taken together, these results suggest that the activation of PAR-2 is involved in cancer invasion and the induction of fibrosis in human pancreatic cancer.
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PMID:Expression of proteinase-activated receptor-2 in human pancreatic cancer: a possible relation to cancer invasion and induction of fibrosis. 1252 25

Protease-activated receptor (PAR)-2 is a G protein-coupled receptor that is activated by trypsin. The purpose of this study was to examine PAR-2 expression and the role of trypsin in cell proliferation in human pancreatic cancer cells. All four pancreatic cancer cell lines studied, from well to undifferentiated types, AsPC-1, BxPC-3, Panc-1, and MIAPaCa-2, had significant levels of PAR-2 mRNA detected by reverse transcription-polymerase chain reaction, and showed a band of about 55 kDa corresponding to the known molecular weight of PAR-2: AsPC-1, BxPC-3 and Panc-1 showed a strong band, and MIAPaCa-2 showed a weak one. Immunocytochemically, AsPC-1, BxPC-3, and Panc-1 showed intense immunostaining for PAR-2, predominantly in the plasma membrane, while in MIAPaCa-2, immunostaining was weak. Proliferative activity of AsPC-1 cells was increased by concentrations of trypsin as low as 10 nM, and activity peaked at a concentration of 100 nM, representing almost 60% of that induced by 10% fetal bovine serum. In contrast, trypsin had no significant effect on proliferation of MIAPaCa-2 cells. These findings suggest that trypsin plays a role in the growth of PAR-2-positive pancreatic cancer cells and serves as a potent mitogen in vitro, functioning as a growth factor.
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PMID:Protease-activated receptor-2 expression and the role of trypsin in cell proliferation in human pancreatic cancers. 1279 76

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.
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PMID:PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors. 1280 82


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