scholarly journals Dual coupling of the α-thrombin receptor to signal-transduction pathways involving phosphatidylinositol and phosphatidylcholine metabolism

1998 ◽  
Vol 337 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Jie CHENG ◽  
Joseph J. BALDASSARE ◽  
Daniel M. RABEN
Keyword(s):  
Signal Transduction ◽  
Cleavage Site ◽  
Thrombin Receptor ◽  
Signal Transduction Pathways ◽  
Metabolizing Enzymes ◽  
Thrombin Cleavage ◽  
Messenger Molecules ◽  
Mitogenic Signalling ◽  
Signalling Cascade ◽  
Phosphatidylcholine Metabolism

Addition of α-thrombin to quiescent IIC9 cells results in the activation of lipid-metabolizing enzymes associated with signal-transduction cascades. These enzymes include phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), phosphatidylcholine (PC)-specific phospholipases C and D and phospholipase A2 (PLA2). Whereas the α-thrombin receptor has been shown to couple with PI-PLCs, it is not clear whether this receptor, or a putative second receptor, couples to one or more of the other phospholipases. In this report we determine whether the cloned receptor couples to all or a subset of these enzymes. We show that (i) an α-thrombin-receptor-activating peptide also elicits the above responses and (ii) addition of enterokinase to IIC9 cells, stably transfected with an α-thrombin receptor (enterokinase- responsive α-thrombin receptor, EKTR) containing an enterokinase cleavage site in place of an α-thrombin cleavage site, stimulates both PI and PC hydrolysis, including PLA2. Enterokinase also induces mitogenesis in the IIC9s transfected with EKTR. These results indicate that, in addition to initiating a mitogenic signalling cascade, the cloned α-thrombin receptor couples to enzymes involved in generating PC-derived, as well as PI-derived, second-messenger molecules in IIC9s. Additionally, using the cells transfected with EKTR, we further demonstrate that only activated, i.e. cleaved, receptors are desensitized.

scholarly journals Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

1992 ◽  
Vol 3 (1) ◽  
pp. 95-102 ◽  
Author(s):  
V Vouret-Craviari ◽  
E Van Obberghen-Schilling ◽  
U B Rasmussen ◽  
A Pavirani ◽  
J P Lecocq ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Synthetic Peptides ◽  
Cleavage Site ◽  
Amino Acid Sequences ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Ccl39 Cells ◽  
G Protein Coupled

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

scholarly journals Stimulation of pancreatic β-cell proliferation by growth hormone is glucose-dependent: signal transduction via Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) with no crosstalk to insulin receptor substrate-mediated mitogenic signalling

1999 ◽  
Vol 344 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Sharon P. COUSIN ◽  
Sigrun R. HülGL ◽  
JR. Martin G. MYERS ◽  
Morris F. WHITE ◽  
Anne REIFEL-MILLER ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Growth Hormone ◽  
Cell Proliferation ◽  
Janus Kinase ◽  
Signal Transduction Pathways ◽  
Insulin Receptor Substrate ◽  
Signal Transducer ◽  
Β Cells ◽  
Β Cell ◽  
Mitogenic Signalling

Mitogenic signal-transduction pathways have not been well defined in pancreatic β-cells. In the glucose-sensitive rat β-cell line, INS-1, glucose (6-18 mM) increased INS-1 cell proliferation (> 20-fold at 15 mM glucose). Rat growth hormone (rGH) also induced INS-1 cell proliferation, but this was glucose-dependent in the physiologically relevant concentration range (6-18 mM glucose). The combination of rGH (10 nM) and glucose (15 mM) was synergistic, maximally increasing INS-1 cell proliferation by > 50-fold. Moreover, glucose-dependent rGH-induced INS-1 cell proliferation was increased further by addition of insulin-like growth factor 1 (IGF-1; 10 nM) to > 90-fold at 12 mM glucose. Glucose metabolism and phosphatidylinositol-3ʹ-kinase (PI3ʹK) activation were necessary for both glucose- and rGH-stimulated INS-1 cell proliferation. Glucose (> 3 mM) independently increased tyrosine-phosphorylation-mediated recruitment of growth-factor-bound protein 2 (Grb2)/murine sons of sevenless-1 protein (mSOS) and PI3ʹK to insulin receptor substrate (IRS)-1 and IRS-2, as well as SH2-containing protein (Shc) association with Grb2/mSOS and downstream activation of mitogen-activated protein kinase and 70 kDa S6 kinase. Glucose-induced IRS- and Shc-mediated signal transduction was enhanced further by the addition of IGF-1, but not rGH. In contrast, rGH was able to activate Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signal transduction at glucose concentrations above 3 mM, but neither glucose independently, nor glucose with added IGF-1, were able to activate the JAK2/STAT5 signalling pathway. Thus rGH-mediated proliferation of β-cells is directly via the JAK2/STAT5 pathway without engaging the Shc or IRS signal-transduction pathways, although activation of PI3ʹK may play an important permissive role in the glucose-dependent aspect of rGH-induced β-cell mitogensis. The additive effect of rGH and IGF-1 on glucose-dependent β-cell proliferation is therefore reflective of rGH and IGF-1 activating distinctly different mitogenic signalling pathways in β-cells with minimal crosstalk between them.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

Abstract The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Signal-regulated oxidation of proteins via MICAL

2020 ◽  
Vol 48 (2) ◽  
pp. 613-620
Author(s):  
Clara Ortegón Salas ◽  
Katharina Schneider ◽  
Christopher Horst Lillig ◽  
Manuela Gellert
Keyword(s):  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Cell Death ◽  
Redox Regulation ◽  
Signal Transduction Pathways ◽  
Cellular Functions ◽  
Intracellular Signals ◽  
Amino Acid Side Chains ◽  
Specific Receptors ◽  
Sulfur Containing

Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions.

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