αA-crystallin and αB-crystallin, newly identified interaction proteins of protease-activated receptor-2, rescue astrocytes from C2-ceramide- and staurosporine-induced cell death

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.