Scaffold Protein SH3BP2 Revealed as Key to Muscle Strength at the Neuromuscular Junction

Every time a muscle contracts, the signal that triggers the movement passes through a microscopic relay station called the neuromuscular junction, where a motor neuron meets a muscle fiber and hands over its chemical message. For decades, researchers have mapped the major players at this synapse, from the acetylcholine receptors that receive the signal to the scaffold proteins that pack them densely into the postsynaptic membrane. Now a team of scientists in Poland and Germany has added an unexpected name to that list: SH3BP2, a scaffold protein previously known mainly for its roles in immune cells and bone, and for causing a rare jaw disorder called cherubism when mutated. In a study published in Frontiers in Molecular Neuroscience, the researchers show that SH3BP2 is a critical organizer of the neuromuscular junction, and that mice lacking the protein in their muscles are measurably weaker because their synapses fall apart under demand.

The story began with alpha-dystrobrevin-1, a cytoplasmic component of the dystrophin-glycoprotein complex, or DGC, a macromolecular assembly that anchors proteins at the muscle membrane to the extracellular matrix and the cytoskeleton. Mutations in several DGC components cause severe, often fatal muscular dystrophies, yet the molecular functions of many of its parts remain incompletely understood. Earlier proteomics work by the same group had identified SH3BP2 as a candidate binding partner recruited to a specific phosphorylated tyrosine residue, Y713, on alpha-dystrobrevin-1. Because SH3BP2 had never been studied in skeletal muscle, the team, led by Tomasz J. Prószyński of Łukasiewicz Research Network–PORT and the Nencki Institute of Experimental Biology, decided to find out what this adapter protein was doing at the synapse.

The first surprise was location. When the researchers immunostained muscle fibers from the tibialis anterior, they found strong SH3BP2 immunoreactivity concentrated precisely at the postsynaptic machinery of the neuromuscular junction, overlapping with the acetylcholine receptors. Electroporating a GFP-tagged version of the protein into living muscle confirmed the enrichment, showing a punctate signal at the junctions where control GFP showed none. Biochemical experiments then established that the interaction with alpha-dystrobrevin-1 is direct and phospho-dependent: synthetic peptides carrying phosphorylated Y713 efficiently pulled down purified SH3BP2, while the unphosphorylated version and a second phosphorylation site, Y730, did not. Domain mapping showed that the C-terminal SH2 domain, a module classically known for binding phosphorylated tyrosines, mediated this specific interaction.

What makes SH3BP2 unusual is its polyvalency. The protein contains three recognizable domains, an N-terminal pleckstrin homology domain, a central proline-rich region, and the SH2 domain, plus a predicted intrinsically disordered region. When the team expressed each domain individually in muscle, all three still targeted the neuromuscular junction, suggesting that each one grips a different postsynaptic partner. Mass spectrometry of proteins pulled down by purified SH3BP2 from cultured myotubes identified several components of the dystrophin-glycoprotein complex, including utrophin, dystrophin, and alpha- and beta-syntrophins, as well as the acetylcholine receptor subunits alpha and gamma and the agrin receptor Lrp4. Co-immunoprecipitation confirmed binding to syntrophin-1 and beta-dystroglycan, the transmembrane core that assembles the entire complex.

The most striking finding, however, concerned the acetylcholine receptor itself. Each receptor pentamer contains two copies of the alpha subunit, and SH3BP2 turned out to bind the long cytoplasmic loop of this subunit at three distinct sites, one for each of its domains. The SH2 domain docked on the most N-terminal segment, the proline-rich region on the next, and the PH domain on the third. Rapsyn, the major receptor-clustering scaffold, bound a completely different, C-terminal set of peptides, meaning the two scaffolds grip the receptor from opposite ends. Purified GST-tagged receptor loop directly captured SH3BP2 from cell extracts, and labeling surface receptors in live myotubes with biotinylated bungarotoxin showed that endogenous SH3BP2 co-precipitates with the receptor complex, confirming the interaction in muscle cells.

To test whether this web of interactions matters functionally, the researchers silenced SH3BP2 in C2C12 myotubes using two independent siRNAs. When the depleted cells were grown on laminin-111 or treated with agrin, both established triggers of receptor clustering, they formed dramatically fewer acetylcholine receptor clusters, with more than a fifty percent reduction under agrin treatment. Crucially, precipitation experiments showed that the total amount of receptor and the amount reaching the cell surface were unchanged. The problem was not receptor production or trafficking but the cell’s ability to gather receptor molecules into dense assemblies at the plasma membrane, pointing to a specific scaffolding defect rather than a general failure of muscle differentiation.

The in vivo experiments extended the story across the entire lifespan of the synapse. The team generated mice lacking SH3BP2 specifically in skeletal muscle by crossing a conditional knockout strain with Myf5-Cre mice. At embryonic day 13.5, before motor axons reach most of the muscle, mutant diaphragms formed significantly fewer of the spontaneously arising receptor hot spots that seed synapse formation, demonstrating a nerve-independent role for the protein. By postnatal day 15, during intense synaptic remodeling, junctions in mutant muscle showed reduced endplate size, reduced receptor-occupied area, and enlarged perforations. In adult animals, the neuromuscular junctions were markedly more fragmented, with an almost fourfold increase in regions where acetylcholinesterase remained exposed after receptors had been removed, a molecular fingerprint of receptor loss from the synapse.

Electron microscopy revealed that the damage ran deeper than the light microscope could see. Normal junctional folds concentrate receptors at their crests and sodium channels in their depths, an arrangement that amplifies the endplate potential into a muscle action potential. In the mutant mice, the folds were exuberantly long and thick, the synaptic cleft was wider, and the electron-dense postsynaptic machinery was often displaced from the crests into the depths of the folds, a compensatory pattern previously seen in animals with reduced rapsyn or lacking alpha-dystrobrevin. These ultrastructural abnormalities help explain why the synapses failed under load: basal transmission was intact, with normal single-pulse endplate potentials and normal compound muscle action potentials at low frequencies, but repetitive stimulation at 50 hertz produced a roughly fifty percent increase in the compound muscle action potential decrement, and single-fiber recordings showed increased fatigue at 20 hertz.

Functional testing tied the synaptic defects directly to weakness. Mutant mice had significantly lower grip strength, ran shorter distances on voluntary running wheels, and exhausted earlier on an accelerating treadmill, reaching lower maximal speeds than controls. Yet histological analysis of muscle cross-sections showed normal fiber morphology and area, and serum creatine kinase levels, a marker of muscle damage, were unchanged. Direct force measurements made the picture precise: when the tibialis anterior muscle was stimulated directly, tetanic force was only mildly reduced, but when the sciatic nerve was stimulated, forcing the signal through the neuromuscular junction, the force deficit became large and significant at every frequency tested. The weakness, in other words, is synaptic, not muscular.

The study positions SH3BP2 as a peripheral member of the dystrophin-glycoprotein complex whose loss disrupts synaptic architecture without triggering muscular dystrophy, mirroring the behavior of syntrophin-1 and utrophin. It also raises intriguing questions beyond the synapse: SH3BP2 was detected in a striated pattern along muscle fibers resembling costameres, the structures that couple contractile machinery to the membrane, and its known association with podosomes in macrophages hints at roles in actin remodeling that the proteomics data partially echo. The authors suggest that future work in aging cohorts and disease models will be needed to determine whether SH3BP2’s contribution to synaptic robustness becomes more consequential as neuromuscular junctions deteriorate with age or disease. For now, the finding adds a new layer to the molecular blueprint of the synapse where brain meets muscle, and identifies a scaffold whose grip on the acetylcholine receptor turns out to be, quite literally, what holds strength together.

Subject of Research: The role of the scaffold protein SH3BP2 in acetylcholine receptor clustering and neuromuscular junction organization in skeletal muscle

Article Title: SH3BP2 deficiency in skeletal muscle leads to neuromuscular junction abnormalities and decreased muscle strength

Article References: Pradhan, B. S., Bernadzki, K. M., Bandaruk, Y., Siudzińska, A., De Cicco, T., Sotomska, M., Gawor, M., Jabłońska, M., Krzemień, J., Chodaczek, G., Hashemolhosseini, S., & Prószyński, T. J. (2026). SH3BP2 deficiency in skeletal muscle leads to neuromuscular junction abnormalities and decreased muscle strength. Frontiers in Molecular Neuroscience, 19, Article 1985823. https://doi.org/10.3389/fnmol.2026.1985823

Image Credits: AI Generated

DOI: 10.3389/fnmol.2026.1985823

Keywords: SH3BP2, neuromuscular junction, acetylcholine receptors, skeletal muscle, dystrophin-glycoprotein complex, alpha-dystrobrevin-1, rapsyn, synaptic transmission, muscle strength, knockout mice, scaffold protein, molecular neuroscience