Identification of spectrin-like repeats required for high affinity utrophin-actin interaction

J Biol Chem. 2005 Jun 17;280(24):23018-23. doi: 10.1074/jbc.M502530200. Epub 2005 Apr 11.

Abstract

Most studies aimed at characterizing the utrophinactin interaction have focused on the amino-terminal tandem calponin homology domain. However, we recently reported evidence suggesting that spectrin-like repeats of utrophin also participate in binding to actin. Here we expressed several recombinant fragments encoding the utrophin amino-terminal domain alone or in combination with various numbers of spectrin-like repeats. We further quantitatively characterized the actin binding properties of each recombinant utrophin fragment using a high-speed sedimentation assay. To evaluate the capacity of each protein to stabilize actin filaments, we compared the effect of utrophin recombinant fragments and full-length utrophin on 6-propionyl-2-(N,N-dimethylamino)naphthalene actin depolymerization. Our results suggest that, whereas the amino-terminal domain is essential for primary interaction between utrophin and actin, spectrin-like repeats have additive effects on the affinity and stoichiometry of binding. Our data indicate that the amino-terminal domain and first 10 consecutive spectrin-like repeats recapitulate the actin binding activity of full-length utrophin more faithfully than the amino-terminal domain alone. These findings support the model for lateral association of utrophin along the actin filament and provide the molecular basis for designing the most effective utrophin "mini-genes" for treatment of dystrophinopathies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / chemistry*
  • Animals
  • Baculoviridae / metabolism
  • Calcium-Binding Proteins / chemistry
  • Calponins
  • Chromatography, Gel
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Epitopes
  • Kinetics
  • Mice
  • Microfilament Proteins
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Spectrin / chemistry*
  • Time Factors
  • Utrophin / chemistry*

Substances

  • Actins
  • Calcium-Binding Proteins
  • Epitopes
  • Microfilament Proteins
  • Recombinant Proteins
  • Utrophin
  • Spectrin