Changes in intracellular localization of calpastatin during calpain activation

Localization of the two main components of the Ca2+-dependent proteolytic s ystem has been investigated in human neuroblastoma LAN-5 cells. Using a mon oclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in t wo granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indic ating that aggregation of calpastatin is a general property and not an excl usive characteristic of neuronal-like cells. The existence of such calpasta tin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounde d organelles. Calpastatin distribution is affected by the intracellular inc rease in free Ca2+, which results in calpastatin progressively becoming a s oluble protein. However, calpain is distributed in the soluble cell fractio n and, in activating conditions, partially accumulates on the plasma membra ne. Similar behaviour has been observed in calpastatin localization in LAN- 5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of, these cells. The involv ement of calpastatin in controlling calpain activity, rather than its activ ation process, and the utilization of changes in calpastatin localization a s a marker of activation of the system is discussed.