Abstract
The unique COOH-terminal domain of dystrophin (mouse dystrophin protein sequences 3266-3678) was expressed as a chimeric fusion protein (with the maltose-binding protein), and its binding to calmodulin was assessed. This fusion protein, called DysS9, bound to calmodulin-Sepharose, bound biotinylated calmodulin, caused characteristic changes in the fluorescence emission spectrum of dansyl-calmodulin, and had an apparent affinity for dansyl-calmodulin of 54 nM. Binding in each case was Ca2+-dependent. The maltose-binding protein does not bind calmodulin, and thus binding resides in the dystrophin-derived sequences. Deletion mutation experiments further localize the high affinity calmodulin binding to mouse dystrophin protein sequences 3293-3349, and this domain contains regions with chemical characteristics found in the calmodulin-binding sequences in other proteins. The COOH-terminal domain provides sites of attachment of dystrophin to membrane proteins, and calmodulin binding may modulate these interactions.
Highlights
Chimeric fusion proteins containing dystrophin amino-terminal domain sequences bind calmodulin (2, 7)
One site phosphorylated by the endogenous activity and by the brain isozyme of CaM kinase II has been localized to dystrophin’s unique COOH-terminal domain (20)
In Becker muscular dystrophy, dystrophin is shortened by genetic deletions, and the regions deleted have been correlated with the severity of disease (23)
Summary
Chimeric fusion proteins containing dystrophin amino-terminal domain sequences bind calmodulin (2, 7). The DGC contains a protein phosphatase activity, which dephosphorylates dystrophin (19), and phosphorylations of dystrophin’s COOH-terminal domain catalyzed by CaM kinase II can be dephosphorylated by calcineurin, the Ca2ϩ-calmodulindependent protein phosphatase type 2b (21). In Becker muscular dystrophy, dystrophin is shortened by genetic deletions, and the regions deleted have been correlated with the severity of disease (23) Deletions that affect this unique COOH-terminal domain result in severe muscular dystrophy, suggesting an important role for this domain in normal muscle function. Since this domain interacts with DGC proteins to anchor dystrophin to the membrane and is phosphorylatable, these protein-protein interactions and their regulation may explain the severe consequences of deletions within the COOH-terminal domain. While it appears that high affinity calmodulin-binding is attributable to these sequences, the site of binding was not where predicted
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