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.
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.
Ubl
J J
JJ
Otto-von-Guericke-Universitat Magdeburg, Medizinische Fakultat, Institut fur Neurobiochemie, Leipziger Strasse 44, 39120 Magdeburg, Germany.
Sergeeva
M
M
Reiser
G
G
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*; D013820:Thermolysin / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology
D008024:Ligands; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; D013820:Thermolysin