Specific role of calpain-1 associated to N-methy l-D-aspartate receptor in lipid rafts microdomains

Although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. Answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. These studies were carried out using human SKNBE neuroblastoma cells. Protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. We have observed that small amounts of calpain-1 are localized in lipid rafts microdomains together with N-methyl-D-aspartate receptor (NMDAR) containing NR1/NR2B subunits. Immunoprecipitation experiments have demonstrated that NMDAR containing NR1/NR2B subunits, calpain-1, HSP90 and neuronal nitric oxide synthase (nNOS) but not calpastatin and calpain-2 are present in specific protein complexes. Thus, in this localization calpain activity is regulated by HSP90 that reduces the affinity for Ca2+ of the protease. Cell stimulation with NMDAR agonists induces calpain activation that specifically cleaves the subunits NR2B of the receptor promoting changes in lipid rafts organization and internalization of NMDAR without affecting cell viability. Moreover, in these conditions, also nNOS is digested and converted in the active form by calpain-1. Our data suggest a physiological role of calpain-1 at specific cell sites. The protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. Following an increase in Ca2+ influx, the activated protease regulated by HSP90, promotes the removal of NMDAR from the plasma membranes, decreasing Ca2+ entrance through this receptor-channel and protecting cells from Ca2+ overloading. The FEBS Journal 283 (Suppl. 1) (2016) 129–417 DOI: 10.1111/febs.13808 187