Protease-activated receptors (PARs) belong to the superfamily of seven transmembrane domain G protein-coupled receptors. Four PAR subtypes are known, PAR-1 to -4. PARs are highly homologous between the species and are expressed in a wide variety of tissues and cell types. Of particular interest is the role which these receptors play in the brain, with regard to neuroprotection or degeneration under pathological conditions. The main agonist of PARs is thrombin, a multifunctional serine protease, known to be present not only in blood plasma but also in the brain. PARs possess an irreversible activation mechanism. Binding of agonist and subsequent cleavage of the extracellular N-terminus of the receptor results in exposure of a so-called tethered ligand domain, which then binds to extracellular loop 2 of the receptor leading to receptor activation. PARs exhibit an extensive expression pattern in both the central and the peripheral nervous system. PARs participate in several mechanisms important for normal cellular functioning and during critical situations involving cellular survival and death. In the last few years, research on Alzheimer's disease and stroke has linked PARs to the pathophysiology of these neurodegenerative disorders. Actions of thrombin are concentration-dependent, and therefore, depending on cellular function and environment, serve as a double-edged sword. Thrombin can be neuroprotective during stress conditions, whereas under normal conditions high concentrations of thrombin are toxic to cells.

Degeneration or survival of cerebral tissue after ischemic injury depends on the source, intensity, and duration of the insult. In the model of focal ischemia, reduced blood flow results in a cascade of pathophysiologic events, including inflammation, excitotoxicity, and platelet activation at the site of injury. One serine protease that is associated closely with and produced in response to central nervous system (CNS) injury is thrombin. Thrombin enters the injury cascade in brain either via a compromised blood-brain barrier or possibly from endogenous prothrombin. Thrombin mediates its action through the protease-activated receptor family (PAR-1, -3, and -4). PARs belong to the superfamily of G protein-coupled receptors with a 7-transmembrane domain structure and are activated by proteolytic cleavage of their N-terminus. We showed that thrombin can be neuroprotective or deleterious when present at different concentrations before and during oxygen-glucose deprivation, an in vitro model of ischemia. We examined the change in mRNA expression levels of PAR-1 to 4 as a result of transient focal ischemia in rat brain, induced by microinjection of endothelin near the middle cerebral artery. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, after ischemic insult on the ipsilesional side, PAR-1 was found to be downregulated significantly, whereas PAR-2 mRNA levels decreased only moderately. PAR-3 was upregulated transiently and then downregulated, and PAR-4 mRNA levels showed the most striking (2.5-fold) increase 12 hr after ischemia, in the injured side. In the contralateral hemisphere, mRNA expression was also affected, where decreased mRNA levels were observed for PAR-1, -2, and -3, whereas PAR-4 levels were reduced only after 7 days. Taken together, these data suggest involvement of the thrombin receptors PAR-1, PAR-3, and PAR-4 in the pathophysiology of brain ischemia.

Protease‐activated receptor‐2 (PAR‐2) is a G protein‐coupled receptor activated by trypsin and other trypsin‐like serine proteases. The widely expressed PAR‐2 is involved in inflammation response but the physiological/pathological roles of PAR‐2 in the nervous system are still uncertain. In the present study, we report novel PAR‐2 interaction proteins, αA‐crystallin and αB‐crystallin. These 20 kDa proteins have been implicated in neurodegenerative diseases like Alexander’s disease, Creutzfeldt‐Jacob disease, Alzheimer’s disease, and Parkinson’s disease. Results from yeast two‐hybrid assay using the cytoplasmic C‐tail of PAR‐2 as bait suggested that αA‐crystallin interacts with PAR‐2. We further demonstrate the in vitro and cellular in vivo interaction of C‐tail of PAR‐2 as well as of full‐length PAR‐2 with αA(αB)‐crystallins. We use pull‐down, co‐immunoprecipitation, and co‐localization assays. Analysis of αA‐crystallin deletion mutants showed that amino acids 120–130 and 136–154 of αA‐crystallin are required for the interaction with PAR‐2. Co‐immunoprecipitation experiments ruled out an interaction of αA(αB)‐crystallins with PAR‐1, PAR‐3, and PAR‐4. This demonstrates that αA(αB)‐crystallins are PAR‐2‐specific interaction proteins. Moreover, we investigated the functional role of PAR‐2 and α‐crystallins in astrocytes. Evidence is presented to show that PAR‐2 activation and increased expression of α‐crystallins reduced C2‐ceramide‐ and staurosporine‐induced cell death in astrocytes. Thus, both PAR‐2 and α‐crystallins are involved in cytoprotection in astrocytes.

Protease-activated receptors (PARs), 7-transmembrane domain G protein-coupled receptors, are involved in tissue degeneration and repair upon injury. We demonstrate the expression of all four PAR subtypes in the postnatal eye and in retina of the adult rat by reverse transcription-polymerase chain reaction (RT-PCR). PAR-1 is regulated developmentally in the eye, with a decrease from P1, P9, to P16, whereas levels for PAR-2, PAR-3, and PAR-4 remain unchanged throughout. In the retina of the adult rat, PAR-1 is highly expressed, whereas PAR-2 and PAR-3 are moderately expressed, compared to low PAR-4 expression. To elucidate possible roles of PARs after trauma, we carried out semiquantitative RT-PCR analysis of expression of all 4 PAR subtypes, beginning 6 hr after partial optic nerve crush (ONC) in the adult rat until 3 weeks after the mild trauma. Levels of PAR mRNA for all four subtypes were upregulated as early as 6 hr after unilateral ONC, except PAR-3, which showed a delayed upregulation. PAR-1, PAR-3, and PAR-4 mRNA levels returned to almost basal levels at 3 weeks post-crush, whereas PAR-2 mRNA level was still high by the end of 3 weeks after crush. Although the lesion was unilateral, PAR mRNA expression in the contralateral, uninjured side was affected to levels almost comparable to those in the injured side. Previous studies have shown an increase in thrombin levels at the site of injury, retinal ganglion cell degeneration by necrosis and apoptosis, and PAR activation as consequences of nerve crush. PAR upregulation because of nerve crush in the mild trauma model could act as an effector of early cell death. Eventual return of receptor mRNA to basal levels is consistent with neuroprotection.

ZusammenfassungThrombin, eine Serinprotease mit zentraler Bedeutung für die Blutgerinnung, kann Rezeptoren auf der Plasmamembran von Zellen aktivieren. Diese Protease-aktivierten Rezeptoren (PARs) wurden kürzlich auch im Gehirn gefunden. Dort sind diese Rezeptoren insbesondere bei Neuroprotektion oder Schädigung im Zusammenhang mit pathologischen Zuständen von Bedeutung. Man unterscheidet 4 Subtypen, die als PAR-1 bis PAR-4 bezeichneten Rezeptoren, die alle in Mensch, Ratte und Maus exprimiert sind und große Homologie zwischen diesen Spezies aufweisen. PARs besitzen einen einzigartigen Aktivierungsmechanismus, weil sie durch die Spaltung des Rezeptorproteins irreversibel in den aktiven Zustand versetzt werden. Das durch die Proteolyse neu entstandene extrazelluläre N-terminale Ende des Rezeptor-Polypeptids wird als ein vorher verborgenes N-terminales Ende offen gelegt und wirkt nun als an das Protein gekoppelter, aktivierender Peptidligand.PARs sind in vielen Bereichen im zentralen und im peripheren Nervensystem exprimiert. PARs scheinen bei der Pathologie neurodegenerativer Erkrankungen, wie Alzheimer oder HIV-Encephalitis eine Rolle zu spielen. Thrombin löst im Gehirn über PARs die Proliferation von Astrozyten aus, was für die reaktive Astrogliose von Bedeutung ist. Andererseits wirkt Thrombin janus-ähnlich, da es bei hohen Konzentrationen Nervengewebe schädigt, dagegen in sehr niedrigen Konzentrationen, welche im leicht geschädigten Nervengewebe erreicht werden, eine protektive Rolle hat.