Sequential degradation of αII and βII spectrin by calpain in glutamate or maitotoxin-stimulated cells

Calpain-catalyzed proteolysis of alpha II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves alpha II spectrin in vitro in repeat 11 between residues Y-1176 and G(1177). Unless stimulated by Ca++ and calmodulin (CaM), beta II spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second alpha II spectrin cleavage at G(1230)*S-1231. beta II spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the beta II spectrin COOH-terminus. CaM promotes beta II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant beta II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alpha II spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alpha II and beta II spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alpha II spectrin cleavage only at Y1176*G1177, while beta II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alpha II and then beta II spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alpha II and beta II spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alpha II spectrin vs beta II spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of beta II spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of beta II spectrin and complete dissolution of the spectrin skeleton.