Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 microM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by any subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 microM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 microM to 20 and 7 microM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.
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PMID:Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand. 867 76

Protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor, is activated when thrombin cleaves its N-terminal exodomain, thereby regulating morphology, growth and survival of neurones and astrocytes. We have investigated the mechanism of PAR-1 desensitisation and resensitisation after proteolytic or non-proteolytic stimulation with thrombin or thrombin receptor agonist peptide (TRag), respectively. In rat primary astrocytes, short-term stimulation with thrombin resulted in a single [Ca2+]i transient and a dose-dependent de- and resensitisation, as assessed by single-cell Ca2+ imaging of fura-2-loaded astrocytes. An initial proteolytic activation of astrocyte PAR-1 by exposure to thrombin strongly decreased the response elicited by subsequent application of a second dose of thrombin or of TRag. In contrast, after an initial non-proteolytic activation of astrocyte PAR-1 by TRag, the subsequent response to thrombin, but not to an additional application of TRag, was strongly attenuated, and the time course for desensitisation was slower. Based on this finding we hypothesised that after PAR-1 activation, the 'tethered ligand' is proteolytically destroyed. As a consequence, the receptor becomes unresponsive to a subsequent thrombin stimulus but is still capable of responding to TRag. This hypothesis was supported by applying thermolysin, which is known to cleave PAR-1 within its tethered-ligand domain, and was confirmed by incubation with soybean trypsin inhibitor. PAR-1 resensitisation occurs via new PAR-1 synthesis since resensitisation was inhibited by cycloheximide and brefeldin A. From these results, we derive a novel model wherein activation of PAR-1, in addition to initiating signal transduction, activates a protease mechanism that cleaves the N-terminus of the receptor, thus terminating the signal and probably inducing receptor internalisation.
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PMID:Desensitisation of protease-activated receptor-1 (PAR-1) in rat astrocytes: evidence for a novel mechanism for terminating Ca2+ signalling evoked by the tethered ligand. 1083 36