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

1. Protease-activated receptor-1. (PAR-1), a G-protein-coupled receptor, is activated when thrombin cleaves its N-terminal exodomain, thereby regulati ng morphology, growth and survival of neurones and astrocytes. We have inve stigated the mechanism of PAR-1 desensitisation and resensitisation after p roteolytic or non-proteolytic stimulation with thrombin or thrombin recepto r agonist peptide (TRag), respectively. 2. In rat primary astrocytes, short-term stimulation with thrombin resulted in a single [Ca2+](i) transient and a dose-dependent de- and resensitisati on, as assessed by single-cell Ca2+ imaging of fura-2-loaded astrocytes. 3. An initial proteolytic activation of astrocyte PAR-1. by exposure to thr ombin strongly decreased the response elicited by subsequent application of a second dose of thrombin or of TRag. In contrast, after an initial non-pr oteolytic activation of astrocyte PAR-1 by TRag, the subsequent response to thrombin, but not to an additional application of TRag, was strongly atten uated, and the time course for desensitisation was slower. 4. Based on this finding we hypothesised that after PAR-1 activation, the ' tethered ligand' is proteolytically destroyed. As a consequence, the recept or becomes unresponsive to a subsequent thrombin stimulus but is still capa ble of responding to TRag. This hypothesis was supported by applying thermo lysin, which is known to cleave PAR-1 within its tethered-ligand domain, an d was confirmed by incubation with soybean trypsin inhibitor. 5. PAR-1 resensitisation occurs via new PAR-1 synthesis since resensitisati on was inhibited by cycloheximide and brefeldin A. 6. From these results, we derive a novel model wherein activation of PAR-1, in addition to initiating signal transduction, activates a protease mechan ism that cleaves the N-terminus of the receptor, thus terminating the signa l and probably inducing receptor internalisation.