Protease activated receptor 2 (PAR-2) belongs to the family of G protein-coupled receptors and is activated by trypsin and tryptase after brain insults. PAR-2 has been implicated in multiple biological functions, such as inflammation, apoptosis, modulation in morphology, proliferation, mitogenesis and immunomodulation. In the current research, we could show earlier that α-crystallin (comprising αA-crystallin and αB-crystallin), a small heat shock protein functioning as structural protein and chaperone, specifically interacts with the PAR-2 protein. Our data indicate that in the PAR family, only PAR-2 interacts with α-crystallin. To understand the interaction of PAR-2 with α-crystallin in detail, mutants of PAR-2 and α-crystallin are used in the experiments. Results from immunoprecipitation and GST pull-down experiments demonstrate that the C-tail of PAR-2 is involved in the interaction with α-crystallin. Specific regions of αA-crystallin (amino acids 120-130 and 136-154) are required for the interaction with PAR-2. Moreover, we investigated the functional role of PAR-2 and α-crystallin in astrocytes. Evidence is presented to show that PAR-2 activation and increased the expression of α-crystallin reduced C2-ceramide- and staurosporine-induced cell death in astrocytes. Thus, both PAR-2 and α-crystallin are involved in cytoprotection in astrocytes. We further investigate the mechanism of protection by PAR-2 and α-crystallin in astrocytes. PAR-2 activation increases expression of αA-crystallin and induces phosphorylation of Ser59 of αB-crystallin. The experiments by mimicking of phosphorylation or unphosphorylation of α-crystallin show that phosphorylation of αA-crystallin at Ser122 and Ser148 and αB-crystallin at Ser45 and Ser59 is required for protection. Ser19 of αB-crystallin has no connection with protection. PAR-2 activation activates p38, ERK and JNK in astrocytes. Application of inhibitors of p38 and ERK reduces the protection by PAR-2 and α-crystallin. Inhibitor of JNK blocks the protection by PAR-2 and does not have any effect on protection by α-crystallin. These findings suggest that PAR-2 and α-crystallin are involved in astrocytes survival by regulating the expression and the phosphorylation status of α-crystallin. In addition, we explored the role of α-crystallin in extracellular matrix. α-Crystallin was expressed in bacteria and purified using chitin resin. Addition of α-crystallin into the extracellular medium rescues astrocytes from staurosporine, C2-ceramide and trypsin treatment. This hints that α-crystallin could be protective in the extracellular medium in neurodegenerative disease conditions.
Protease-activated receptor-2, α-crystallin, protein interaction, astrocytes, small heat shock protein, phosphorylation, neuropr