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.