Utrophin Lacks the Rod Domain Actin Binding Activity of Dystrophin

Abstract

We previously identified a cluster of basic spectrinlike repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419 -28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI ‫؍‬ 4.86) lacks the basic character of the repeats found in DYSR11-17 (pI ‫؍‬ 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly ␣-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K d of 7.3 ؎ 1.3 M and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 M. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.

Quadriceps myopathy caused by skeletal muscle-specific ablation of βcyto-actin

“…These deletions result in a truncated dystrophin that lacks fully functional spectrinlike repeats 17 and 18. Previous studies indicated that spectrinlike repeat 17 is localized to the second actin binding domain of dystrophin (Amann et al, 1999) and is also important for anchoring neuronal nitric oxide synthase (nNOS) to the sarcolemma (Lai et al, 2009). Moreover, both spectrin-like repeat 17 and 18 can bind anionic phospholipids (Legardinier et al, 2009).…”

“…The actin-filament binding activity of spectrin-like repeat 17 (Amann et al, 1999) combined with the fact that dystrophin binds filamentous actin composed of different actin isoforms with similar affinities (Renley et al, 1998) led us to hypothesize that spectrinlike repeat 17 might also interact with  cyto -actin. Although we did not detect  cyto -actin on peeled sarcolemma (Rybakova et al, 2000), another study showed sarcolemmal localization of  cytoactin on an in situ skeletal muscle preparation (Lubit and Schwartz, 1980).…”

Chloride channel activity of ClC-2 is modified by the actin cytoskeleton

“…Furthermore, ClC-2 does not possess typical actin-binding motifs such as those present in actinin. However, the present studies in itro suggest that the N-terminus of ClC-2 can interact with actin (K d 0.8 µM) with an affinity for actin similar to that of dystrophin [23]. Further, because the interaction between the N-terminus peptide and F-actin is modified by ionic strength, the interaction might occur via electrostatic interactions.…”

“…Further, the acidic C-terminus of band 3 can interact specifically with carbonic anhydrase [27]. Moreover, dystrophin, the membrane protein that is defective in Duchenne muscular dystrophy, has been shown to interact with actin via a series of basic repeats within its rod domain ; this interaction is sensitive to increasing ionic strength [23]. This interaction is thought to be important for normal arrangement of the actin cytoskeleton.…”

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer

“…24,25 One difference between them is that the rod domain actin-binding site of dystrophin is not present in utrophin. 26 Nevertheless, Rybakova et al 27 showed that overexpression of utrophin in mdx muscle rescued the defective link between costameric actin filaments and the sarcolemma. They concluded that, even if molecular epitopes differ between the two proteins, utrophin and dystrophin seem to be functionally interchangeable actin-binding proteins.…”