The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation
| Autor: | C L Potter, K J Norton, Wadie F. Bahou, M S Goligorsky, Barry S. Coller, Jeffery L. Kutok |
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| Rok vydání: | 1993 |
| Předmět: |
Blood Platelets
Umbilical Veins Platelet Aggregation Molecular Sequence Data Peptide Receptors Cell Surface CHO Cells Biology Ligands Thrombin Cricetinae Thrombin receptor medicine Animals Humans Receptor chemistry.chemical_classification Base Sequence Chinese hamster ovary cell General Medicine Fusion protein Molecular biology Peptide Fragments Recombinant Proteins Amino acid Biochemistry chemistry Immunoglobulin G Human umbilical vein endothelial cell Calcium Receptors Thrombin Endothelium Vascular medicine.drug Research Article |
| Zdroj: | The Journal of clinical investigation. 91(4) |
| ISSN: | 0021-9738 |
| Popis: | A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s). |
| Databáze: | OpenAIRE |
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