| Calcium dependent neutral proteases (calpain) have been demonstrated to participate in long-term synaptic plasticity. To identify the critical downstream events, we studied the effects of calpain on several proteins that play important roles in synaptic function.;Calpain-mediated proteolysis of GRIP and PSD-95 was first studied in vitro by calpain I digestion of rat forebrain membranes. In both cases, several degradation products were identified suggesting the existence of multiple cutting sites. Most of the fragments were later confirmed following in situ calpain activation by brief NMDA stimulation of cultured hippocampal slices, which indicates that calpain-mediated proteolysis of GRIP and PSD-95 might occur in vivo. We also found the PSD-95 breakdown products were abundant in developing hippocampus, and dramatically declined during the adulthood. Combined with previous reports, we proposed that calpain degradation of PSD-95 is involved in synaptic remodeling during development, and possibly in adulthood following appropriate stimulus. To explore the potential functions of calpain-mediated GRIP degradation, we analyzed GRIP and GluR2 interaction using co-immunoprecipitation techniques. We found that calpain activation resulted in the disruption of GRIP-GluR2 interaction and since GRIP has been hypothesized as AMPA receptor anchoring protein, such disruption could facilitate release of the AMPA receptors from the synaptic sites.;To further address the impact of calpain activation on glutamate receptor trafficking, frozen-thawed rat forebrain sections were first treated with calcium to activate endogenous calpain, and PSD proteins were extracted as Triton-insoluble fractions. Following calpain activation, AMPA receptor subunits GluR1 and GluR2 are fragmented and, interestingly, the degradation species of the subunits were absent from PSDs and recovered in Triton-soluble fractions. Our data thus revealed the potential function of calpain in glutamate receptor trafficking.;The study on calpain degradation of alpha-CaMKII suggested that autophosphorylated form of alpha-CaMKII is resistant to calpain-mediated proteolysis. Since it has been previously shown that calpain decreases the activity of non-phosphorylated form of the kinase, our results thus argue that calpain might function to decrease the kinase activity.;In summary, our studies demonstrated the regulatory role of calpain on several important synaptic molecules and provided biochemical evidence for calpain involvement in synaptic modification. |
