RESTRICTION OF MU-M-CALCIUM-REQUIRING CALPAIN ACTIVATION TO THE PLASMA-MEMBRANE IN HUMAN NEUROBLASTOMA-CELLS - EVIDENCE FOR REGIONALIZED INFLUENCE OF A CALPAIN ACTIVATOR PROTEIN

Regulation of the mu M-calcium-requiring form of calpain (mu calpain) was studied in SH-SY-5Y human neuroblastoma cells, Immunoblot analysis demonstrated that the vast majority of mu calpain is localized within cytosolic pools, Calpain activation was monitored as a function of au tolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact n euronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, In, calpain is preferen tially activated at the plasma membrane as evidenced by autolytic gene ration of faster-migrating isoforms, By contrast, similar autolytic pr ofiles for mu calpain in membrane or cytosolic fractions following add ition of calcium were observed under cell-free conditions and within c ells following death due to extended ionophore-mediated calcium influx , These differential activation profiles for cytosolic mu calpain with in living cells and following cellular fractionation/cell death indica te the presence of a regulatory system within neuronal cells, As in pr evious studies in other systems, we demonstrate the presence of a calp ain activator protein, Cycloheximide treatment depleted the autolytic capacity of membrane-associated mu calpain within 4-6 hr without a cor responding decline in total mu calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpa in; pulse-chase radiolabeling confirmed the half-life of mu, calpain t o exceed 24 hr, Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuron al cells, and that, given the tremendous latent mu calpain activity wi thin the cytosol, the interplay of the activator protein and the endog enous inhibitor calpastatin are crucial for maintaining neuronal homeo stasis.