Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.Study of calpain 3 received an important impetus after the demonstration of its involvement in limb girdle muscular dystrophy type 2A (Mendelian Inheritance in Man [MIM] 253600) (24). This neuromuscular disorder is characterized mainly by symmetrical atrophy and weakness of proximal limb muscles, by elevated creatine kinase in serum, and by a dystrophic pattern in muscle biopsies (4). Calpains are members of a family of intracellular nonlysosomal cysteine proteases (for reviews, see references 35 and 36). They are comprised of three ubiquitous calpains (, m, and /m); a skeletal musclespecific calpain (calpain 3, CAPN3, nCL-1, or p94 [31]), a variant of which is also expressed in a lens-specific manner (17, 18); a digestive tract-specific calpain ); and the stomach-specific calpains (nCL-2 and nCL-2Ј [33]).The human calpain 3 gene was reported to consist of 24 exons spanning approximately 45 kb (24). It encodes a 3.5-kb mRNA expressed predominantly in skeletal muscle tissues. The 821-amino-acid-long calpain 3 protein can be subdivided, like the other calpains, into four domains that include a proteolytic (domain II) and a calcium binding (domain IV) domain (26, 31). In addition, three short calpain 3-specific sequences (NS, IS1, and IS2 [36]) are present. These are located, respectively, at the N terminus, in the protease domain, and between domains III and IV. IS2 includes a titin (connectin) binding site (11, 34) as well as a putative nuclear localization signal (31). Calpain 3 differs from the ubiquitous calpains by its rapid autolysis, at least when it is expre...