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Calpain-catalyzed proteolysis of 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 II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII 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 II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while 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 alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII 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 betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.

Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet the functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alphaII spectrin cDNA from human fetal brain (GenBank and ). On the basis of the predicted amino acid sequence, 11 amino acid substitutions, presumably representing polymorphisms, have been identified that distinguish this alphaII spectrin from human lung fibroblast alphaII spectrin. In addition, human fetal brain spectrin displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung fibroblast alphaII++ spectrin. This discovery, together with two previously identified regions of alternative mRNA splicing in alphaII spectrin suggest that as many as eight different splice forms of the mature protein might exist if all combinations (at inserts 1, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of alternative exon usage was investigated by semiquantitative PCR with intron-jumping primer sets. Tissues examined were from mouse and included heart, kidney, lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, as well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within alphaII spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the novel 15 bp insertion reported here, were only expressed in brain, heart, skeletal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, even under conditions in which a 30% level of insert 3 negative transcript could be easily detected in artificially prepared control samples. All combinations of insert 1 and insert 2 were identified together in individual transcripts, verifying at least four distinct isoforms of alphaII spectrin. These have been named alphaIISigma1 through alphaIISigma4, in accord with current spectrin naming conventions. Dynamic molecular modeling of the 15th repeat unit incorporating insert 2 predicts that the spliced sequence forms a loop between helices A and B, and suggests that this insert might constitute a novel protein interaction site. The presence of this sequence in alphaIISigma3 and alphaIISigma4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)