Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16–17

Abstract

Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the β-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.

First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene

“…In striated skeletal and cardiac muscle, a full-length transcript of 14 kb encodes dystrophin, a subsarcolemmal 427 kDa rod-shaped protein incorporated into a multimolecular membrane-associated protein complex that connects intracellular cytoskeleton to extracellular matrix. This complex ensures membrane stability during muscle contraction and mediates cellular signaling [ 1 , 2 ]. The dystrophin protein is defective in Duchenne muscular dystrophy (DMD), the most severe and frequent muscular dystrophy in children [ 3 ].…”

Perspective: Spectrin-Like Repeats in Dystrophin Have Unique Binding Preferences for Syntrophin Adaptors That Explain the Mystery of How nNOSμ Localizes to the Sarcolemma

“…The recent study by Adams et al (2018) may finally provide the answer to this mystery. They performed a search for novel SBS in dystrophin, focusing on dystrophin’s spectrin-like repeat region and amino terminus.…”

“… Adams et al (2018) then demonstrated that peptides containing the putative SBS in spectrin-like repeats 17, 22, but not 23, could pull down syntrophin from skeletal muscle homogenates. Importantly, spectrin-like repeats 17 and 22 had unique syntrophin isoform preferences.…”

“…The model is controversial because of equivocal evidence for the ability of dystrophin to bind nNOSμ directly ( Lai et al, 2013 ). A recent study by Adams et al (2018) , suggests a new model that may finally solve the mystery of the dual requirement for dystrophin and α-syntrophin for nNOSμ localization to the sarcolemma ( Figure 1 ). The purpose of this commentary is to highlight this new model for sarcolemmal nNOSμ localization and to discuss its implications in the context of ongoing clinical testing of the ability of miniaturized dystrophins to mitigate dystrophic pathology in individuals with DMD.…”

“…Therefore, a major function of syntrophins is to recruit signaling proteins to the dystrophin protein complex. To identify novel binding sites, Adams et al (2018) identified a new SBS consensus sequence using known SBS in dystrophin and related proteins from evolutionarily close and distant species. The SBS consensus sequence was a 14 amino acid long α-helix with a strictly conserved histidine at position zero.…”