Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.Posttranslational modification of proteins is an important mechanism for the regulation of cellular processes. Our understanding is perhaps most advanced for modifications that involve protein phosphorylation and dephosphorylation. By contrast, we know relatively little about the enzymes that catalyze intracellular proteolytic cleavage and about the biological roles of these cleavage reactions. Proteases that mediate the activation or inactivation of proteins during and after export from cells have been studied extensively, but the enzymes involved in similar events in the cytoplasm have not been identified in most cases. Calpains are one class of proteins that may serve such a function. They are intracellular proteases which are active at neutral pHs, and their activity is regulated by calcium concentration and by specific inhibitors and activators (8,16,19,41,46). Among their potential substrates are proteins such as transcription factors, hormone receptors, protein kinases, and phosphatases, as well as components of the cytoskeleton (8,16,53).In vertebrates, four different classes of calpain have been characterized so far on the sequence level. The two major calpains, calpain I and calpain II, are heterodimeric proteins with a large subunit and a small subunit (45). The large subunit consists of four domains; domain II is related to other thiol proteases, and domain IV is calmodulin-like, with several calcium-binding EF-hand motifs. Domains I and III show no similarity to other known proteins. The small subunit is common to calpain I and calpain II. It has two domains, a hydrophobic N-terminal glycine-rich domain and a C-terminal calmodulin-like domain which is closely related to that in the large subunit. Calpains I and II differ in their calcium requirement; calpain...

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