The carboxyterminal EF domain of erythroid α-spectrin is necessary for optimal spectrin-actin binding

Spectrin and protein 4.1R crosslink F-actin, forming the membrane skeleton. Actin and 4.1R bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF domain, is calmodulin-like, with calcium-dependent and calcium-independent EF hands. The severely anemic sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF domain, implying that the domain is critical for skeletal integrity. To test this, we constructed a minispectrin heterodimer from the actin-binding domain, the EF domain, and 4 adjacent spectrin repeats in each chain. The minispectrin bound to F-actin in the presence of native human protein 4.1R. Formation of the spectrin-actin-4.1R complex was markedly attenuated when the minispectrin contained the shortened sph(1J) alpha-spectrin. The alpha-spectrin deletion did not interfere with spectrin heterodimer assembly or 4.1R binding but abolished the binary interaction between spectrin and F-actin. The data show that the alpha-spectrin EF domain greatly amplifies the function of the beta-spectrin actin-binding domain (ABD) in forming the spectrin-actin-4.1R complex. A model, based on the structure of alpha-actinin, suggests that the EF domain modulates the function of the ABD and that the C-terminal EF hands (EF34) may bind to the linker that connects the ABD to the first spectrin repeat. (Blood. 2010;116(14):2600-2607)