Dystrophin-Glycoprotein Complex Is Monomeric and Stabilizes Actin Filaments in Vitro through a Lateral Association

Inna N Rybakova,James M Ervasti

doi:10.1074/jbc.272.45.28771

Abstract

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (Mr 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind G-actin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophin-glycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes F-actin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.

Highlights

Skeletal muscle dystrophin can be purified as part of a large oligomeric complex containing integral and peripheral sarcolemmal proteins, several of which are glycoproteins [1,2,3]
We did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin, which suggests that these proteins can simultaneously bind to an actin filament
The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization was dependent on the dystrophin-glycoprotein complex concentration and saturated at a dystrophin:actin ratio of 0.04 corresponding to 1 dystrophin/25 actin monomers (Fig. 2B). This result is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin measured at equilibrium [15] and suggests that dystrophin binding to F-actin is responsible for the protective effect of dystrophinglycoprotein complex on actin depolymerization

Summary

Introduction

Skeletal muscle dystrophin can be purified as part of a large oligomeric complex containing integral and peripheral sarcolemmal proteins, several of which are glycoproteins [1,2,3]. We did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin, which suggests that these proteins can simultaneously bind to an actin filament. These results suggest that dystrophin exists as a monomer in the glycoprotein complex and binds alongside an actin filament with 0.5 ␮M affinity via two or more discrete low affinity binding sites distributed throughout its amino-terminal and rod-like domains.

Results

Conclusion