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MS 0327 Received 15 November 1999; accepted after revision 8 March 2000.
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ABSTRACT |
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INTRODUCTION |
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There is increasing recent evidence that members of the serine protease family, including thrombin and trypsin, serve as signalling molecules via activation of specific protease-activated receptors (PARs), evoking a variety of cellular responses (for review, see Déry et al. 1998). So far, four members of this new receptor family have been identified: PAR-1, the prototype of the new family of G-protein-coupled receptors, which is activated by thrombin; PAR-2, which is activated by trypsin and mast cell tryptase (Nystedt et al. 1994; Molino et al. 1997; Corvera et al. 1999); and PAR-3 and PAR-4, which are activated by thrombin (Vu et al. 1991; Ishihara et al. 1997; Xu et al. 1998). Proteases cleave PARs within the N-terminal exodomain. This cleavage creates a new N-terminus, constituting a tethered-ligand domain, which binds intramolecularly to a site within the same receptor molecule to induce transmembrane signalling (Rasmussen et al. 1991; Vu et al. 1991; Gerszten et al. 1994; Nanevicz et al. 1995). This concept has been confirmed by experiments using synthetic peptides that mimic the tethered-ligand domain. These peptides function as receptor agonists, activating the receptor as would a peptide hormone, i.e. without proteolytic cleavage (Vu et al. 1991; Chen et al. 1994).
In the brain the serine proteases play a pivotal role in normal and pathological conditions, affecting both neuronal and glial cells (Grand et al. 1996; Turgeon & Houenou, 1997). In vitro studies revealed that thrombin stimulates DNA synthesis, induces proliferation, and causes inhibition of stellation and rounding of astrocytes in culture (Cavanaugh et al. 1990; Beecher et al. 1994). Thrombin can protect astrocytes and neurones from cell death after environmental insults (Vaughan et al. 1995) and can attenuate neuronal cell death and modulate astrocyte reactivity induced by the 
-amyloid peptide in vitro (Pike et al. 1996). However, depending on the concentration used, thrombin can also induce apoptosis in astrocytes and some types of neurone (Donovan et al. 1997; Smirnova et al. 1998; Turgeon et al. 1998). Since most of the cellular responses in astrocytes and neurones described so far for thrombin can also be induced by thrombin receptor-activating peptides (TRAPs) and since the mRNA for PAR-1 has been identified within the brain (Weinstein et al. 1995), it is generally accepted that thrombin exerts its cellular effects in the brain via activation of PAR-1.
Generally, cellular responses transmitted by activation of G-protein-coupled receptors are rapidly attenuated. Mechanisms of signal attenuation include removal of agonist from the extracellular fluid, receptor desensitisation, receptor endocytosis and receptor downregulation (for review, see Böhm et al. 1997). However, after a certain time period the cellular response recovers and cells resensitise. These important processes determine the ability of cells to respond to agonists. Desensitisation prevents the uncontrolled long-lasting stimulation of cells, and resensitisation allows cells to recover or maintain their responsiveness. Therefore, it is of great interest to understand the mechanisms that modulate receptor signalling. It is a special challenge to understand these processes for thrombin-evoked cellular responses since in contrast to other G-protein-coupled receptors, the thrombin receptor is activated by an irreversible protein modification through proteolytic cleavage, generating a tethered ligand within the body of the receptor. Despite the irreversible nature of the proteolytic cleavage, signalling by PARs is rapidly terminated.
The question of how signalling after activation of the receptor by thrombin is shut off has been extensively studied in endothelial cells, fibroblasts and blood-derived cell lines. These studies have shown that desensitisation of PAR-1 is due to receptor phosphorylation by G-protein-coupled receptor kinases or second messenger kinases and/or activation-triggered internalisation of the receptor (Hoxie et al. 1993; Hein et al. 1994; Ishii et al. 1994; Woolkalis et al. 1995; Shapiro et al. 1996). However, in endothelial cells a significant fraction of the receptors remain on the surface or are recycled, although they do not respond to thrombin, nor do they self-activate (Woolkalis et al. 1995). In megakaryoblastic CHRF 288 cells, PAR-1 can be reactivated by thrombin if the receptor is recycled at 4°C but not at 37°C (Brass et al. 1994). Thrombin-cleaved platelet receptors are not internalised and remain on the surface in a desensitised state (Norton et al. 1993); and thrombin-desensitised fibroblasts expressing wildtype PAR-1 do not respond to a second application of thrombin but do respond to the TRAP SFLLRN (Hammes & Coughlin, 1999), suggesting that mechanisms other than phosphorylation and endocytosis may regulate PAR-1. As a possible explanation for the presence of non-self-activating receptors in the membrane, it has been suggested that the N-terminal ligand may be altered or hidden within a protected environment (Norton et al. 1993; Greco et al. 1996).
Despite the apparent importance of thrombin-mediated signalling in brain physiology and pathology, the question of PAR-1 desensitisation and resensitisation has not yet been assessed in detail in brain-derived cells. In previous reports from our laboratory, we have shown that in primary cultures of rat astrocytes, short-term (60 s) stimulation with thrombin or thrombin receptor agonist peptide (TRag) induces a dose-dependent Ca2+ response and that long-term stimulation resulted in a prolonged, biphasic Ca2+ response or [Ca2+]i oscillations in more than half of the cells examined. After washout of thrombin or upon simultaneous superfusion with the endogenous thrombin inhibitor protease nexin-1, [Ca2+]i returned to pre-stimulation levels and re-addition of the protease or removal of the inhibitor elicited an attenuated Ca2+ response (Ubl & Reiser, 1997; Ubl et al. 1998). These data clearly demonstrate that despite the irreversible nature of receptor activation, dissociation of thrombin from its receptor rapidly terminates the Ca2+ signal and induces desensitisation, therefore leaving the cells less responsive to a further challenge with the protease.
The purpose of the present study was to elucidate the mechanisms of PAR-1 signal termination and de- and resensitisation in rat astrocytes. We have investigated the Ca2+ response elicited by repeated application of thrombin and TRag. We found that PAR-1 activation most probably induces the activity of a trypsin-like protease that destroys the tethered-ligand domain. This conclusion gives rise to a novel mechanism for the termination of PAR-1 transmembrane signalling and triggering of receptor de- and resensitisation.
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METHODS |
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Materials
Thrombin (human), thermolysin, soybean trypsin inhibitor (SBTI) and cycloheximide were from Sigma. The cell culture medium was from Gibco/BRL except for fetal calf serum, penicillin and streptomycin, which were from Biochrom (Berlin, Germany). Fura-2 AM was from Molecular Probes. The synthetic thrombin receptor agonist peptide (TRag) with the sequence Ala-4-fluoro-L-Phe-Arg--cyclohexyl-L-Ala-L-homoArg-Tyr-NH2 was from Neosystems Laboratoire (Strasbourg, France). Brefeldin A, thapsigargin and cytochalasin D were from Calbiochem.
Cell culture
Primary astrocyte-enriched cell cultures were obtained according to the method of Hamprecht & Löffler (1985). All experiments conformed to guidelines from Sachsen-Anhalt on the ethical use of animals and all efforts were made to minimise the number of animals used. In brief, newborn rats were decapitated, and the brains were removed and collected in ice-cold Puck's D1 solution (mM: 137·0 NaCl, 5·4 KCl, 0·2 KH2PO4, 0·17 Na2HPO4, 5·0 glucose and 58·4 sucrose, pH 7·4). The brains were gently passed through nylon meshes of 250 and 136 µm pore width, in consecutive order. The cell suspension was centrifuged at 4°C for 5 min at 500 g. The cells were resuspended in 10 ml growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % (v/v) fetal calf serum, 20 U ml-1 penicillin and 20 µg ml-1 streptomycin). The cells were plated on round coverslips (22 mm diameter) in culture dishes (50 mm diameter) at a density of 3-5 × 105 cells per dish and incubated at 37°C with 10 % CO2, humidified to saturation. The medium was changed after 5 days and thereafter every 2-3 days depending on the cell density. For experiments, cells were used between days 6 and 14 in culture.
Cytosolic Ca2+ measurements
The free cytosolic Ca2+ concentration ([Ca2+]i) was measured using the Ca2+-sensitive fluorescent dye fura-2. For dye loading, the cells grown on a coverslip were placed in 1 ml Hepes-buffered saline (HBS; buffer composition (mM): 145 NaCl, 5·4 KCl, 1 MgCl2, 1·8 CaCl2, 25 glucose and 20 Hepes, pH 7·4 adjusted with Tris) supplemented with 2 µM fura-2 AM, for 30 min at 37°C. Loaded cells were transferred into a perfusion chamber with a bath volume of about 0·2 ml and mounted on an inverted microscope (Zeiss, Axiovert 135). During the experiments the cells were continuously superfused with medium heated to 37°C. The perfusion system was combined with a six-port valve (Thomachrom, Type RH 0112; Reichelt, Heidelberg, Germany) to allow switching between solutions containing the different agents to be tested.
Single-cell fluorescence measurements of [Ca2+]i were performed using an imaging system from T.I.L.L. Photonics GmbH (Munich, Germany). Cells were excited alternately at 340 and 380 nm for 25-75 ms at each wavelength with a rate of 0·33 Hz, and the resultant emission was collected above 510 nm. Images were stored on a computer, and subsequently the fluorescence ratio (F340/F380) was determined from selected regions of interest covering single cells. Results are expressed as means ± S.E.M. Differences between two groups of data were examined by Student's t test, with P < 0·05 considered to be significant.
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RESULTS |
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PAR-1 desensitisation and resensitisation
In rat astrocytes, short-term activation of PAR-1 with thrombin or TRag elicited a transient increase of [Ca2+]i. The amplitude of the transient response induced strongly depended on the agonist concentration (Ubl et al. 1998). To determine the characteristics of PAR-1 desensitisation, rat astrocytes were stimulated twice for 60 s with two different concentrations of thrombin, generating submaximal (0·01 U ml-1) and maximal (0·1 U ml-1) responses. Between the two stimuli the cells were washed for 5 min with HBS. Representative single-cell measurements obtained with 0·01 and 0·1 U ml-1 thrombin are shown in Fig. 1A and B, respectively. Repeated short-term activation of PAR-1 elicited separate [Ca2+]i transients; the amplitude of the second transient was attenuated, indicative of receptor desensitisation. For quantification, the amplitudes of the transients were calculated as the maximum change in fluorescence ratio (
F340/F380), and the response to the second stimulus was given as a relative response (percentage of initial response), i.e. in relation to the mean amplitude of the first transient. The size of the first response was concentration dependent, with absolute amplitudes (F340/F380) of 0·511 ± 0·039 (n = 99) and 0·839 ± 0·019 (n = 521) for 0·01 and 0·1 U ml-1 thrombin, respectively. Similarly, the attenuation of the second thrombin-induced [Ca2+]i transient showed concentration dependence. The amplitude of the second [Ca2+]i transient, given as a percentage of the intitial response, was 63 ± 5·2 % (n = 99) for 0·01 U ml-1 thrombin and 32 ± 2·0 % (n = 211) for 0·1 U ml-1 thrombin.
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Rat astrocytes were stimulated by a brief pulse (60 s) of 0·01 U ml-1 thrombin (A) or 0·1 U ml-1 thrombin (B). After 5 min of washing in HBS, the cells were again stimulated with thrombin at the same concentration for 60 s, and after another 5 min of washing they were challenged with 10 µM ATP for 30 s, as indicated by the respective bars. The traces in A and B represent typical single-cell measurements from rat astrocytes loaded with fura-2. The results were confirmed in four and three individual experiments, respectively, with at least 20 single cells measured. C, time course of resensitisation obtained for either 0·01 U ml-1 thrombin ( | ||
By increasing the time interval between the two stimuli, the cells regained their ability to respond to thrombin. Again, the time course of resensitisation depended on the agonist concentration, as shown in Fig. 1C. With 0·01 U ml-1 thrombin the cells recovered within 15 min, whereas with 0·1 U ml-1 thrombin, after 30 min the response recovered to about 70 % of the first response and up to 2 h were required for full recovery. In order to prove that the reduced response to a second challenge was due to desensitisation of PAR-1 and not to an incomplete refilling of intracellular stores after the first stimulation, we compared the thapsigargin-induced release of Ca2+ from intracellular stores 5 min after a prior stimulation with thrombin and, as a control, without thrombin pretreatment. As shown in Fig. 2, the amount of Ca2+ released by thapsigargin was not altered by a preceding challenge with 0·1 U ml-1 thrombin. Additionally, we observed that the amplitude of a subsequent Ca2+ response induced by 10 µM ATP was independent of the amplitude of the previous response to thrombin (Fig. 1A and B).
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To evaluate the refilling of intracellular Ca2+ stores after short-term thrombin stimulation, the Ca2+ content of the stores was estimated by addition of 1 µM thapsigargin (Tg) 5 min after a preceding thrombin application. As a control, rat astrocytes were challenged with 1 µM thapsigargin without prior thrombin stimulation. The traces are representative for results obtained from 14 and nine single cells from two experiments with (continuous line) and without (dashed line) prior thrombin activation, respectively. | ||
Mechanism of PAR-1 desensitisation
The fact that PARs are activated by a proteolytic mechanism suggests that, after cleavage, PARs should remain activated. However, both our present results obtained with short-term stimulation and our previous results using long-term stimulation of astrocytes (Ubl & Reiser, 1997) showed that the Ca2+ signal persisted only as long as the agonist remained present; after washout of the agonist the Ca2+ signal rapidly terminated. To elucidate the underlying mechanism of desensitisation, we took advantage of the property of PAR-1 that the receptors can be activated not only proteolytically by thrombin but also non-proteolytically by receptor-activating peptides. We used TRag, a synthetic thrombin receptor agonist peptide, since it is more potent (Feng et al. 1995) and has higher selectivity for PAR-1 over PAR-2 (Kawabata et al. 1999) compared with the natural thrombin receptor-activating peptide SFLLRN. We examined the Ca2+ responses evoked by repeated homologous stimulation with either thrombin (0·1 U ml-1) or TRag (1 µM) with an intervening wash of 5 min with HBS, and heterologous stimulation with both agonists. The concentrations of thrombin and TRag employed here induce nearly maximal responses in rat astrocytes, as assessed in our previous work (Ubl et al. 1998). The mean amplitude of the first TRag-induced [Ca2+]i transient was, at 0·678 ± 0·016 (n = 443), slightly but significantly smaller than that induced by thrombin. Therefore, the response to a second PAR-1 activation was always compared with the mean amplitude of the first [Ca2+]i transient caused by the same agonist. The responses measured in astrocytes exposed sequentially to thrombin or TRag are summarised in Table 1.
Table 1. The [Ca2+]i response in rat astrocytes induced by repeated stimulation with agonists
| First agonist | Response ( F340/F380) |
Second agonist | Response ( F340/F380) |
Desensitisation (%) |
| Thrombin (0·1 U ml-1) | 0·84 ± 0·019 | Thrombin (0·1 U ml-1 ) | 0·26 ± 0·016 | 68·6 |
| TRag (1 µM) | 0·26 ± 0·02 | 61·7 | ||
| TRag (1 µM) | 0·68 ± 0·016 | TRag (1 µM) | 0·45 ± 0·034 | 33·1 |
| Thrombin (0·1 U ml-1 ) | 0·31 ± 0·025 | 63·4 |
F340/F380) was measured in individual cells. Experiments were repeated on at least three different cultures, with > 10 cells examined per culture. Percentage desensitisation was calculated by comparing the second response with the initial response to the same agonist.
Irrespective of the activation mode, the second stimulation of PAR-1 always led to an [Ca2+]i transient of diminished amplitude. However, the degree of attenuation depended on the agonists used and on the sequence of their addition. After a proteolytic activation of PAR-1 with thrombin, the subsequent Ca2+ responses to thrombin or TRag were strongly attenuated to a similar extent (almost 70 %; see Table 1). However, the data obtained with an initial non-proteolytic PAR-1 activation gave an unexpected result, as shown in Table 1. After non-proteolytic activation we observed only moderate desensitisation (decrease of 33 %) in the subsequent response to TRag, but a strong attenuation (decrease of 63 %) of the amplitude of the Ca2+ response that could be elicited with thrombin as the second agonist. The most plausible explanation for this difference observed after TRag activation is that TRag not only activates PAR-1, but also alters and thereby inactivates the tethered-ligand domain. As a consequence, the receptor would be unresponsive to thrombin but still responsive to TRag.
One possible way to 'silence' the tethered-ligand domain might be to cleave the N-terminus within this domain. We indirectly tested our hypothesis using thermolysin, a metalloprotease known to cleave PAR-1 within the tethered-ligand domain at amino acid positions Phe43/Leu44 and Leu44/Leu45 (Chen et al. 1996), resulting in a thrombin-insensitive receptor. Figure 3 shows representative averaged response traces obtained from rat astrocytes superfused with thermolysin (5 U ml-1)followed by thrombin (0·1 U ml-1; Fig. 3A) or TRag (1 µM; Fig. 3B) for 60 s. Superfusion of rat astrocytes with thermolysin for 90 s did not alter the resting [Ca2+]i. However, thermolysin completely inhibited the Ca2+ response evoked by a subsequent addition of 0·1 U ml-1 thrombin, whereas the response to TRag was not altered. The results illustrated in Fig. 3A and B are summarised in Fig. 3C. These data clearly demonstrate that a prior cleavage of the tethered-ligand domain of PAR-1 caused an inhibition of the proteolytic, but not the non-proteolytic, receptor activation pathway.
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Rat astrocytes were superfused with 5 U ml-1 thermolysin for 90 s prior to the addition of 0·1 U ml-1 thrombin (A) or 1 µM TRag (B), as indicated by the respective bars. The traces shown are the mean responses measured from n single cells in a single experiment. C, summary of the maximum change of the fluorescence ratio ( | ||
In addition, we wanted to assess whether cleavage, and thus covalent modification, of the receptor might influence the desensitisation. Consequently, we examined the time course for recovery of the second response with homologous and heterologous stimulation. Figure 4 shows the relative responses (percentage of initial response) caused by a second stimulation for 60 s with either 0·1 U ml-1 thrombin or 1 µM TRag obtained at different time points after the initial thrombin (Fig. 4A) or TRag (Fig. 4B) challenge (60 s). Interestingly, in both cases the non-proteolytic primary activation of PAR-1 induced a slower time course of desensitisation than did the proteolytic primary activation. The fact that the proteolytic activation resulted in a faster desensitisation of the Ca2+ response indicates that cleavage of the receptor may serve as a signal for rapid receptor desensitisation.
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The responses obtained by applying a second pulse for 60 s of either 0·1 U ml-1 thrombin ( | ||
Based on these results, we used thermolysin as a model protease for an inactivating cleavage of PAR-1. We added thermolysin directly before or after the initial thrombin pulse and examined the response to a second PAR-1 stimulation 5 min after the first one. As Table 2 summarises, thermolysin, given before or after the first thrombin addition, inhibited the response to a second thrombin application by nearly 100 %. As expected, giving thrombin prior to thermolysin application caused a strong attenuation (by 75 %) of the subsequent response to TRag, similar to the attenuation obtained with thrombin alone (69 % in Table 1). However, even when avoiding an initial activation of PAR-1 by superfusion with thermolysin prior to thrombin, the response to a subsequent addition of TRag was reduced to 72 % of the initial response. This result implies that even non-activating cleavage of the N-terminus of PAR-1 by thermolysin was sufficient to induce receptor desensitisation to TRag.
Table 2. Influence of PAR-1 cleavage, induced by thermolysin before or after an initial stimulation with thrombin, on a second Ca2+ response induced either by thrombin or by TRag
| Second agonist |
Thermolysin application | |
| Before thrombin (% initial response) |
After thrombin (% initial response) |
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| Thrombin (0·1 U ml-1 ) | 0 (18) | 5 ± 3·5 (13) |
| TRag (1 µM) | 72 ± 11·7 (26) | 25 ± 3·2 (44) |
Recently it was shown that platelet activation with the PAR-1-activating peptide SFLLRN was associated with cleavage of PAR-1 and the release of a peptide that could be detected by antibodies directed against the 41 amino acid residue peptide released by thrombin activation of PAR-1 (Ofosu et al. 1998). This SFLLRN-induced PAR-1 cleavage could be prevented by the serine protease inhibitor 4,2-(aminoethyl)-benzene sulphonylfluoride-HCl (pefabloc SC) and soybean trypsin inhibitor (SBTI), but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, the authors concluded that a trypsin-like protease propagates SFLLRN-dependent PAR-1 cleavage. These findings are compatible with our hypotheses and, consequently, we conducted similar experiments. Rat astrocytes were preincubated for 90 s with SBTI (100 ng ml-1) and stimulated for 60 s with TRag. This was followed by a 5 min wash with HBS supplemented with SBTI and a subsequent challenge with thrombin or TRag. Preincubation with SBTI did not alter the resting [Ca2+]i and exerted no influence on an initial thrombin or TRag response (data not shown). However, as summarised in Fig. 5, the presence of SBTI significantly increased the response to TRag and thrombin obtained 5 min after non-proteolytic PAR-1 activation compared with the controls (5 min superfusion without SBTI). These results using rat astrocytes confirm the findings of Ofosu and co-workers (Ofosu et al. 1998), i.e. that stimulation of PAR-1 induces the activation of a trypsin-like protease. Furthermore, these data support our hypothesis that a proteolytic cleavage of the tethered-ligand domain of PAR-1 leaves the receptor less capable of responding to a subsequent challenge with thrombin compared with the response to TRag.
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Rat astrocytes were preincubated for 90 s with 100 ng ml-1 SBTI prior to stimulation with 1 µM TRag. Subsequently the cells were washed for 5 min in the presence of SBTI and then stimulated for a second time either with 0·1 U ml-1 thrombin or with 1 µM TRag. The response to the second stimulus, given as a percentage of the initial response to the same agonist, is shown as the mean ± S.E.M. of (n) single cells for SBTI ( | ||
Mechanism of PAR-1 resensitisation
As shown in Fig. 1C, after a recovery period of 30 min the response to a second activation of PAR-1 with 0·1 U ml-1 thrombin resensitised to about 70 % of the initial response. To study the possible mechanisms of PAR-1 resensitisation further, we incubated cells during the recovery period (30 min) with 50 µg ml-1 brefeldin A (to disturb the integrity of the Golgi apparatus), 100 µM cycloheximide (to inhibit protein synthesis), or 20 µM cytochalasin D (to disrupt the cytoskeleton). Importantly, the control responses evoked by thrombin were not affected by preincubation for 30 min with any of these drugs (data not shown). Incubation of astrocytes with cytochalasin D caused dramatic changes in cellular morphology. This was, however, without any effect on the de- and resensitisation process of the protease-evoked response (Table 3). In contrast, disturbing the Golgi apparatus with brefeldin A and inhibition of protein biosynthesis with cycloheximide significantly attenuated the recovery of the response to thrombin (Table 3), indicating that receptor synthesis and translocation to the membrane are prerequisites for resensitisation.
Table 3. Influence of different compounds on resensitisation of the thrombin-induced Ca2+ response
| Wash (30 min) | Response to thrombin (% initial response) |
| Control buffer | 71 ± 3·3 (133) |
| Cytochalasin D (20 µM) | 65 ± 4·6 (33) |
| Brefeldin A (50 µg ml-1) | 24 ± 4·0 (33) |
| Cycloheximide (100 µM) | 33 ± 4·0 (46) |
Additionally, we checked the influence of protein biosynthesis on resensitisation after homologous or heterologous stimulation with thrombin and TRag. Figure 6 shows that inhibition of protein biosynthesis with cycloheximide differentially influenced the recovery of the response to thrombin or to TRag. Independent of the activation mechanism involved in the first stimulus, only the subsequent thrombin-elicited response was substantially attenuated in the presence of cycloheximide; in contrast, the magnitude of a second response induced by TRag was independent of new protein biosynthesis.
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After an initial stimulation with 0·1 U ml-1 thrombin (Thr) or 1 µM TRag, cells were incubated for 30 min in HBS either without ( | ||
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DISCUSSION |
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The current work was undertaken to characterise the mechanism of PAR-1 desensitisation and resensitisation in rat astrocytes. We have previously characterised the PAR-1-evoked Ca2+ responses in rat astrocytes (Ubl & Reiser, 1997; Ubl et al. 1998). We showed that the amplitude of the thrombin- or thrombin receptor activating-peptide (TRag or SFLLRN)-elicited Ca2+ signal was concentration dependent and that removal of the agonists resulted in an immediate and rapid cessation of the Ca2+ signal induced. Therefore, the change in [Ca2+]i can be used as a physiological parameter to investigate the regulation of PAR-1 signalling.
The unique activation mechanism of PARs by specific proteolysis of the receptor implies that the receptors become irreversibly activated. Therefore, mechanisms have to exist that shut off the protease-induced signals. Well-characterised mechanisms for G-protein-coupled receptor signal termination and desensitisation include receptor phosphorylation and internalisation. Such mechanisms seem to be involved in PAR-1 regulation since phosphorylation of the thrombin receptor was shown to inhibit PAR-1 signalling in a number of different cells (Brass, 1992; Ishii et al. 1994; Selak, 1994; Weihong et al. 1998). Using differential antibody binding or by studying tagged receptors, it was additionally shown that activation-triggered internalisation of PAR-1 is responsible for the signal termination and desensitisation in the megakaryoblastic cell lines HEL and CHRF 2, human endothelial cells (Hoxie et al. 1993; Brass et al. 1994; Woolkalis et al. 1995), and the human neuronal cell line Ad12 HER 10 (Weinstein et al. 1998).
At first sight, our results demonstrating that after proteolytic cleavage of PAR-1 the subsequent responses to thrombin or TRag were reduced to the same extent indicate that also in rat astrocytes receptor phosphorylation and/or internalisation might be responsible for the signal attenuation observed. However, our further finding, that after non-proteolytic activation of PAR-1 the subsequent Ca2+ response to thrombin was strongly attenuated while the TRag-elicited Ca2+ transient was only moderately decreased, convincingly indicates that an additional mechanism for signal termination might exist. If activation-triggered receptor phosphorylation and/or internalisation, resulting in the uncoupling of the receptors from G-proteins and the disappearance of the receptor from the cell surface, respectively, are the sole processes that terminate PAR-1-induced Ca2+ signals, then the degree of attenuation of the response to a subsequent stimulus should be similar for the two agonists, irrespective of a possible rapid recycling of a modified receptor.
Based on our present results, we propose a modified model for the mechanism responsible for termination of PAR-1 transmembrane signalling and for PAR-1 de- and resensitisation (Fig. 7). Our findings suggest an alternative, new mechanism for termination of PAR-1-induced Ca2+ signalling. In rat astrocytes, stimulation of PAR-1 seems to induce the activation of a trypsin-like serine protease, which destroys the tethered-ligand domain of PAR-1. As a consequence, the proteolytically elicited PAR-1 signal is shut off.
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Activation of PAR-1 is evoked either by proteolytic cleavage when thrombin binds to the receptor via the hirudin-like domain (left side of the diagram) or by a non-proteolytic mechanism triggered by reversible binding of TRag to the ligand-binding domain (right side of the diagram). Additionally, an as yet unidentified trypsin-like protease is activated, which cleaves the receptor after washout of the agonists, within or C-terminal to the tethered-ligand domain, thereby terminating transmembrane signalling by the tethered ligand. Furthermore, proteolytic activation of PAR-1 triggers a rapid phosphorylation/internalisation, leaving the cells unresponsive to both thrombin and TRag. With artificial non-proteolytic activation, phosphorylation/internalisation proceeds on a slower time course. The ability of a cell subsequently to respond to thrombin strongly depends on new synthesis and insertion of intact PAR-1 receptors into the membrane, whereas a fraction of the receptors can recycle and maintain responsiveness to TRag. Activation of PAR-1 involves phospholipase C activity, generating InsP3 and diacylglycerol (DAG). | ||
An enigmatic mechanism for shut-off and desensitisation, specific for PAR-1, has already been hypothesised by other groups based on the following findings. Firstly, analysis of the cloned platelet thrombin receptor with a polyclonal antibody recognising the cleavage region revealed that thrombin cleaves and activates the receptor. However, this did not initiate redistribution or internalisation of the receptor (Norton et al. 1993). Nevertheless, pretreatment of platelets with thrombin led to the loss of binding of this antibody and the disappearence of the cellular shape change induced by thrombin, providing evidence that receptor cleavage is followed by the loss of the N-terminal peptide (tethered ligand). Secondly, in CHRF 288 cells a significant fraction of PAR-1 receptors are recycled back to the plasma membrane after activation and internalisation, but these receptors are not responsive to a further challenge with thrombin, nor do they self-activate at 37°C, as would be expected if the tethered ligand were still present (Brass et al. 1994). However, in this study it was shown that the receptors could be reactivated if they were recycled at 4°C. The authors concluded that after activation, a structural change of PAR-1 takes place that does not occur at 4°C (based on our results, this might be through the action of a proteolytic enzyme). Finally, in studies using fibroblasts from PAR-1 knock-out mice, which regained their responsiveness to either thrombin or the thrombin receptor agonist peptide SFLLRN when the cells were transfected with human PAR-1 cDNA, it has been observed that, when desensitised to thrombin, these fibroblasts could still respond to SFLLRN but not to thrombin (Hammes & Coughlin, 1999). All of these results suggest that other mechanisms besides phosphorylation or endocytosis may regulate PAR-1.
In summary, these points of evidence from the literature and our own results reported herein imply that modification of the tethered ligand may be involved in the termination of thrombin signalling. An interesting hint as to the nature of such a mechanism comes from recent work using human platelets (Ofosu et al. 1998) showing that SFLLRN not only activates but also induces cleavage of PAR-1 by the action of a trypsin-like protease. Consequently, we hypothesise that an additional proteolytic cleavage modifies and thus inactivates the tethered-ligand domain of PAR-1. Such a mechanism could also explain previous findings by Norton et al. (1993), Brass et al. (1994) and Hammes & Coughlin (1999). Furthermore, our finding that thermolysin, known to cleave PAR-1 within and C-terminal to the tethered-ligand domain (Chen et al. 1996), inhibits the response to a subsequent proteolytic, but not non-proteolytic, activation of PAR-1 in astrocytes clearly demonstrates that an incapacitating cleavage of PAR-1 leaves the cell unresponsive to subsequent proteolytic activation, thus supporting our hypothesis of the existence of a novel mechanism for terminating the Ca2+ signalling of PAR-1. In addition, the experiments with thermolysin indicate that a non-activating cleavage of PAR-1 is sufficient to induce desensitisation of the TRag-evoked PAR-1 response, although to a lesser extent than an activating cleavage.
The strongest evidence for an incapacitating proteolytic cleavage of PAR-1 after receptor activation comes from our experiments showing that in the presence of SBTI, the degree of desensitisation to thrombin after an initial TRag challenge was significantly reduced. However, SBTI also reduced the desensitisation to the peptide itself. This can be explained by the fact that a non-activating cleavage of the N-terminus of the receptor results in desensitisation, as found for treatment with thermolysin (see Table 2). This desensitisation most probably implies receptor phosphorylation/internalisation. As a consequence, inhibition of the non-activating cleavage by SBTI also alters the desensitisation to TRag. The mechanism proposed here is also supported by recent work showing that plasmin, an anticoagulant protease, desensitised the thrombin receptor via a specific cleavage in the linker region between the tethered-ligand domain and the ligand-binding domain (Kuliopulos et al. 1999).
The question arises as to why we could not observe the proposed inactivating cleavage after proteolytic activation of PAR-1. One explanation could be that proteolytic activation of the receptor leaves the receptor insensitive to a second thrombin stimulus but capable of responding to TRag. However, we found that after an initial challenge with the protease, the subsequent responses to thrombin and TRag were diminished to the same extent (see Table 1). Investigation of the time course of de- and resensitisation of the PAR-1-induced Ca2+ response in rat astrocytes revealed a slower time course for desensitisation following non-proteolytic activation. This difference might reflect the fact that an activating cleavage and the concomitant conformational change of the receptor serve as a trigger for a more rapid internalisation of the receptor and/or receptor phosphorylation, therefore leaving the cells unresponsive to thrombin and also to TRag.
Similar to the situation in HEL cells (Brass et al. 1991) and CHRF 288 cells (Hoxie et al. 1993), resensitisation of the thrombin response strongly depends on new receptor synthesis and translocation of PAR-1 to the plasma membrane, as assessed with cycloheximide and brefeldin A. The time period of 30 min for cycloheximide-sensitive recovery of the thrombin response is short. This high sensitivity is indicative of a high turnover rate of PAR-1 in cultured rat astrocytes. Our finding that the recovery of the TRag response was not significantly influenced by cycloheximide implies that a sizeable fraction of the internalised receptors are recycled back to the plasma membrane. The fact that these receptors can be activated by TRag but not by thrombin lends support to our novel model wherein an additional cleavage after receptor activation prevents self-activation of PAR-1. However, modifications of the receptor that occur during internalisation and recycling, leaving the receptor insensitive to thrombin but still responsive to TRag, cannot be excluded.
Taken together, we have strong evidence that further degradation (proteolysis) of the tethered-ligand domain is involved in PAR-1 signal termination, preventing self-activation of the receptor after removal of thrombin, as depicted in Fig. 7. The cleavage site and identity of the responsible protease remain to be elucidated. Furthermore, it is necessary to find out whether this mechanism is specific for PAR-1 or whether this is generally true for PAR signal attenuation, since such a mechanism has strong implications for pharmacological intervention in PAR signalling. It is exciting to speculate that similar mechanisms might apply even to some G-protein-coupled receptors that are activated by agonists other than proteases.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Beecher, K. L., Anderson, T. T., Fenton, J. W. II & Festoff, B. W. (1994). Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture. Journal of Neuroscience Research 37, 108-115 | [Medline] |
| Böhm, S. K., Grady, E. F. & Bunnett, N. W. (1997). Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochemical Journal 322, 1-18 | [Medline] |
| Brass, L. F. (1992). Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation. Journal of Biological Chemistry 267, 6044-6050 | [Abstract] |
| Brass, L. F., Manning, D. R., Williams, A. G., Woolkalis, M. J. & Poncz, M. (1991). Receptor and G protein-mediated responses to thrombin in HEL cells. Journal of Biological Chemistry 266, 958-965 | [Abstract] |
| Brass, L. F., Pizarro, S., Ahuja, M., Belmonte, E., Blanchard, N., Stadel, J. M. & Hoxie, J. A. (1994). Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling. Journal of Biological Chemistry 269, 2943-2952 | [Abstract] |
| Cavanaugh, K. P., Gurwitz, D., Cunningham, D. D. & Bradshaw, R. A. (1990). Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1. Journal of Neurochemistry 54, 1735-1743 | [Abstract] |
| Chen, J., Ishii, M., Wang, L., Ishii, K. & Coughlin, S. R. (1994). Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. Journal of Biological Chemistry 269, 16041-16045 | [Abstract] |
| Chen, X., Earley, K., Luo, W., Lin, S.-H. & Schilling, W. P. (1996). Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand. Biochemical Journal 314, 603-611 | [Medline] |
| Corvera, C. U., Déry, O., McConalogue, K., Gamp, P., Thoma, M., Al-Ani, B., Caughey, G. H., Hollenberg, M. D. & Bunnett, N. W. (1999). Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2. The Journal of Physiology 517, 741-756 | [Abstract/Full Text] |
| Déry, O., Corvera, C. U., Steinhoff, M. & Bunnett, N. W. (1998). Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. American Journal of Physiology 43, C1429-1452. | |
| Donovan, F. M., Pike, C. J., Cotman, C. W. & Cunningham, D. D. (1997). Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. Journal of Neuroscience 17, 5316-5326 | [Abstract/Full Text] |
| Feng, D. M., Veber, D. F., Connolly, T. M., Condra, C., Tang, M. J. & Nutt, R. F. (1995). Development of a potent thrombin receptor ligand. Journal of Medicinal Chemistry 38, 4125-4130 | [Medline] |
| Gerszten, R. E., Chen, J., Ishii, M., Ishii, K., Wang, L., Nanevicz, T., Turck, C. W., Vu, T. K. & Coughlin, S. R. (1994). Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 368, 648-651 | [Medline] |
| Grand, R. J. A., Turnell, A. S. & Grabham, P. W. (1996). Cellular consequences of thrombin-receptor activation. Biochemical Journal 313, 353-368 | [Medline] |
Greco, N. J., Jones, G. D., Tandon, N. N., Kornhauser, R., Jackson, B. & Jamieson, G. A. (1996). Differentiation of two forms of GPIb functioning as receptors for  -thrombin and von Willebrand factor: Ca2+ responses of protease-treated human platelets activated with -thrombin and the tethered ligand peptide. Biochemistry 23, 915-921. |
|
| Hammes, S. R. & Coughlin, S. R. (1999). Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: Evidence for a novel mechanism for preventing or terminating signaling by PAR 1's tethered ligand. Biochemistry 38, 2486-2493 | [Medline] |
| Hamprecht, B. & Löffler, F. (1985). Primary glial cultures as a model for studying hormone action. Methods in Enzymology 109, 341-345 | [Medline] |
| Hein, L., Ishii, K., Coughlin, S. R. & Kobilka, B. K. (1994). Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. Journal of Biological Chemistry 269, 27719-27726 | [Abstract] |
| Hoxie, J. A., Ahuja, M., Belmonte, E., Pizarro, S., Parton, R. & Brass, L. F. (1993). Internalization and recycling of activated thrombin receptors. Journal of Biological Chemistry 268, 13756-13763 | [Abstract] |
| Ishihara, H., Connolly, A. J., Zeng, D., Kahn, M. L., Zheng, Y. W., Timmons, C., Tram, T. & Coughlin, S. R. (1997). Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386, 502-506 | [Medline] |
| Ishii, K., Chen, J., Ishii, M., Koch, W. J., Freedman, N. J., Lefkowitz, R. J. & Coughlin, S. R. (1994). Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases. Journal of Biological Chemistry 269, 1125-1130 | [Abstract] |
| Kawabata, A., Saifeddine, M., Al-Ani, B., Leblond, L. & Hollenberg, M. D. (1999). Evaluation of proteinase-activated receptor-1 (PAR(1)) agonists and antagonists using a cultured cell receptor desensitization assay: Activation of PAR(2) by PAR(1)-targeted ligands. Journal of Pharmacology and Experimental Therapeutics 288, 358-370 | [Abstract/Full Text] |
| Kuliopulos, A., Covic, L., Seeley, S. K., Sheridan, P. J., Helin, J. & Costello, C. E. (1999). Plasmin desensitization of the PAR-1 thrombin receptor: Kinetics, sites of truncation, and implications for thrombolytic therapy. Biochemistry 38, 4572-4585 | [Medline] |
| Molino, M., Barnathan, E. S., Numerof, R., Clark, J., Dreyer, M., Cumashi, A., Hoxie, J. A., Schechter, N., Woolkalis, M. & Brass, L. F. (1997). Interactions of mast cell tryptase with thrombin receptors and PAR-2. Journal of Biological Chemistry 272, 4043-4049 | [Abstract/Full Text] |
| Nanevicz, T., Ishii, M., Wang, L., Chen, M., Chen, J., Turck, C. W., Cohen, F. E. & Coughlin, S. R. (1995). Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site. Journal of Biological Chemistry 270, 21619-21625 | [Abstract/Full Text] |
| Norton, K. J., Scarborough, R. M., Kutok, J. L., Escobedo, M. A., Nannizzi, L. & Coller, B. S. (1993). 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 82, 2125-2136 | [Abstract] |
| Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994). Molecular cloning of a potential proteinase activated receptor. Proceedings of the National Academy of Sciences of the USA 91, 9208-9212 | [Medline] |
| Ofosu, F. A., Freedman, J., Dewar, L., Song, Y. & Fenton, J. W. II (1998). A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation. Biochemical Journal 336, 283-285 | [Medline] |
| Pike, C. J., Vaughan, P. J., Cunningham, D. D. & Cotman, C. W. (1996). Thrombin attenuates neuronal cell death and modulates astrocytes reactivity induced by -amyloid in vitro. Journal of Neurochemistry 66, 1374-1382. |
[Abstract] |
| Rasmussen, U. B., Vouret-Craviari, V., Jallat, S., Schlesinger, Y., Pages, G., Pavirani, A., Lecocq, J.-P., Pouyssegur, J. & Van Oberghen-Schilling, E. (1991). cDNA cloning and expression of a hamster -thrombin receptor coupled to Ca2+ mobilization. FEBS Letters 288, 123-128 |
[Medline] |
| Selak, M. A. (1994). Cathepsin G and thrombin: evidence for two different platelet receptors. Biochemical Journal 297, 269-275 | [Medline] |
| Shapiro, P. S., Evans, J. N., Davis, R. J. & Posada, J. A. (1996). The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases. Journal of Biological Chemistry 271, 5750-5754 | [Abstract/Full Text] |
| Smirnova, I. V., Zhang, S. X., Citron, B. A., Arnold, P. M. & Festoff, B. W. (1998). Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons. Journal of Neurobiology 36, 64-80 | [Medline] |
| Turgeon, V. L. & Houenou, L. J. (1997). The role of thrombin-like (serine) proteases in the development, plasticity and pathology of the nervous system. Brain Research Reviews 25, 85-95. | [Medline] |
| Turgeon, V. L., Lloyd, E. D., Wang, S., Festoff, B. W. & Houenou, L. J. (1998). Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. Journal of Neuroscience 18, 6882-6891 | [Abstract/Full Text] |
| Ubl, J. J. & Reiser, G. (1997). Characteristics of thrombin-induced calcium signals in rat astrocytes. Glia 21, 361-369 | [Medline] |
| Ubl, J. J., Vöhringer, C. & Reiser, G. (1998). Co-existence of two types of calcium-inducing protease-activated receptors (PAR-1 and PAR-2) in rat astrocytes and C6 glioma cells. Neuroscience 86, 597-609 | [Medline] |
| Vaughan, P. J., Pike, C. J., Cotman, C. W. & Cunningham, D. D. (1995). Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults. Journal of Neuroscience 15, 5389-5401 | [Abstract] |
| Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. (1991). Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057-1068 | [Medline] |
| Weihong, Y., Tiruppathi, C., Lum, H., Qiao, R. & Malik, A. (1998). Protein kinase C regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells. American Journal of Physiology 274, C387-395 |
[Medline] |
| Weinstein, J. R., Gold, S. J., Cunningham, D. D. & Gall, C. M. (1995). Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. Journal of Neuroscience 15, 2906-2919 | [Abstract] |
| Weinstein, J. R., Lau, A. L., Brass, L. F. & Cunningham, D. D. (1998). Injury-related factors and conditions down-regulate the thrombin receptor (PAR-1) in a human neuronal cell line. Journal of Neurochemistry 71, 1034-1050 | [Abstract] |
| Woolkalis, M. J., DeMelfi, T. M. Jr, Blanchard, N., Hoxie, J. A. & Brass, L. F. (1995). Regulation of thrombin receptors on human umbilical vein endothelial cells. Journal of Biological Chemistry 270, 9868-9875 | [Abstract/Full Text] |
| Xu, W.-F., Andersen, H., Whitmore, T. E., Presnell, S. R., Yee, D. P., Ching, A., Gilbert, T., Davie, E. W. & Foster, D. C. (1998). Cloning and characterization of human protease-activated receptor 4. Proceedings of the National Academy of Sciences of the USA 95, 6642-6646 | [Abstract/Full Text] |
This work was supported in part by the INTAS Programme (grant 94-3852), Deutsche Forschungsgemeinschaft (436 RUS/17/24/98), LSA (grant 2923A) and Fonds der Chemischen Industrie. We thank Regina Wender for critical reading and help with the manuscript.
Corresponding author
G. Reiser: Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Institut für Neurobiochemie, Leipziger Straße 44, D-39120 Magdeburg, Germany.
Email: georg.reiser{at}medizin.uni-magdeburg.de
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) or 0·1 U ml-1 thrombin (
). The amplitude of the second response is given as a percentage of the initial response obtained after the interval indicated on the time scale. The values are means ± S.E.M. of at least 20 single cells from at least two different preparations.
) and with (
) prior cleavage of PAR-1 by thermolysin. The data shown are the means ± S.E.M. of (n) single cells from at least two different preparations.