Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure

Abstract

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.

Keywords: AChR clustering; MuSK phosphorylation; congenital myasthenic syndromes; dimerization; muscle-specific kinase (MuSK); neuromuscular junction; receptor tyrosine kinases; β2-adrenergic agonists.

Figure 1

The NMJ and the agrin‐LRP4‐MuSK‐DOK7 pathway. (a) Schematic representation of the NMJ and the agrin‐LRP4‐MuSK‐DOK7 pathway. (b) The binding of agrin to the N‐terminal region of LRP4 induces a conformational change (active state) promoting the binding between LRP4 and MuSK first IgG‐like domain (Zhang, Coldefy, Hubbard, & Burden, 2011). This results in MuSK activation via dimerization and trans‐autophosphorylation of tyrosine residues within the cytoplasmic region (Schlessinger, 2000) through a not fully known mechanism. The increase in the catalytic activity creates active binding sites for other proteins such as DOK7, which amplifies the signal downstream. LRP4 is composed of eight low‐density lipoprotein Class A (LDLa) domains (blue) at the N‐terminus, followed by four YWTD β‐propeller domains (gray) bounded by epidermal growth factor‐like modules (yellow) and a short C‐terminal domain (Springer, 1998). LRP4 self‐associates and interacts with MuSK in the absence of agrin (inactive state), but is not capable of activating MuSK (Kim et al., 2008). This interaction could be important for AChRs prepatterning before innervation (Yang et al., 2001; Yumoto et al., 2012). IgG, immunoglobulin G; LRP, low‐density lipoprotein receptor‐related protein; MuSK, muscle‐specific receptor tyrosine kinase; NMJ, neuromuscular junction

Figure 2

MuSK domains structure and conservation. (a) Schematic representation of MuSK drawn to linear scale and location of variants found in the patient (bold) and mutations previously reported in the literature (black). MuSK (NP_005583.1) is composed of three IgG‐like domains, a FzD, a short transmembrane domain (TMD), a JMR, a KD, and a short C‐terminal tail. (b) Protein alignments were performed using the ClustalW2 multiple sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). (c) Segregation analysis of MUSK variants in the family. Pedigree symbols are shaded according to the presence of clinical symptoms. FzD, frizzled domain; IgG, immunoglobulin G; JMR, juxtamembrane region; KD, kinase domain; MuSK, muscle‐specific receptor tyrosine kinase

Figure 3

Molecular modeling of MuSK variants. (a) Cartoon representation of MuSK Frizzled‐like domain structure (MMDB ID: 76272) with two copies of the asymmetric dimmer colored in green and blue. Structural elements are labeled in white text according to (Stiegler, Burden, & Hubbard, 2009) and the p.Cys317 residue in red text. (b) Cartoon representation of MuSK KD monomer structure (MMDB ID: 20673). Structural elements are labeled according to (Till et al., 2002). MuSK KD structure is made of β‐strands and α‐helices (green). MuSK activity is regulated by the juxtamembrane domain at the N‐terminus (αB) and the activation loop (αAL; pink). The catalytic loop (CL) is represented in red color. The αC‐helix is colored gray and the p.Ala617 residue is labeled in red. The tyrosine residues within the activation loop (Tyr‐750/754/755) are shown in stick representation (blue). The crystal structure of the MuSK KD after dimerization is not known. (c) The residue p.Cys317 forms a disulfide bond with p.Cys381. Modeling of p.C317R shows how the substitution of cysteine to an arginine result in the disruption of an essential disulfide bond leading to multiple clashes with the neighboring residues. (d) The p.Ala617 residue (red color) is located in the αC‐helix at the N‐terminal lobe of the MuSK KD. Using the crystal structure by (Till et al., 2002), the substitution of alanine to valine does not result in changes in distance and/or a clash with other residues. KD, kinase domain; MuSK, muscle‐specific receptor tyrosine kinase

Figure 4

Effect of MuSK variants on AChR clustering. (a) The retroviral infection of C2C12 MusK KO recovered successfully MuSK protein expression and AChR clustering function. Bar graphs show the quantification of AChR clusters from MuSK WT and variants. Data points represent the mean data from 40 images for each condition per experiment (n = 4). One‐way ANOVA and Dunnet's multiple comparisons test (*p < .05; ***p < .001). (b) Quantification of average AChR cluster size, total AChR cluster area per field of vision (FOV), and raw integrated density in MuSK WT and p.A617V myotubes. Data points represent the mean data from 40 images for each condition per experiment (n = 4). Two‐tailed unpaired t test (***p < .01). ANOVA, analysis of variance; KO, knock‐out; MuSK, muscle‐specific receptor tyrosine kinase; WT, wild‐type

Figure 5

Effect of MuSK variants on protein expression, stability, and localization. (a) MuSK WT and p.A617V expressed correctly at the cell surface of HEK‐293T cells after PEI transfection of WT and mutant pMUSK‐mCherryN1 constructs, while MuSK p.C317R remained mostly cytoplasmic (green arrows). Cell nuclei were stained with DAPI. (b) Pull‐down of MuSK p.C317R from the cell surface of HEK‐293T was reduced to 19.43 ± 8.78% compared with WT. The results (ratio IP: WCL) are represented as mean ± SEM from n = 3 experiments. One‐way ANOVA and Dunnett's multiple comparisons test (**p < .01). ANOVA, analysis of variance; DAPI, 4′,6‐diamidino‐2‐phenylindole; IP, immunoprecipitation; PEI, polyethylenimine; MuSK, muscle‐specific receptor tyrosine kinase; SEM, standard error of mean; WCL, whole cell lysates; WT, wild‐type

Figure 6

Effect of p.A617V on MuSK tyrosine phosphorylation in HEK‐293T cells. (a) MuSK tyrosine phosphorylation was increased in the p.A617V variant (*) compared with WT following incubation with full‐length agrin for one hour. (b) MuSK tyrosine phosphorylation was also significantly increased in the p.A617V variant (*) in the absence of agrin incubation as shown by the densitometry analysis. Results are shown as mean ± SEM relative to WT from n = 5 experiments. Two‐tailed unpaired t test (p < .01). Lanes 1 and 2 are loading controls while lanes 5–9 are controls to ensure the specificity of the pull‐down assay. EGFP, enhanced green fluorescent protein; IP, immunoprecipitation; MuSK, muscle‐specific receptor tyrosine kinase; SEM, standard error of mean; WCL, whole cell lysates; WT, wild‐type

Figure 7

Effect of p.A617V on baseline MuSK kinase activity and agrin‐induced temporal activation profile in muscle cells. (a) In the absence of agrin, there was increased phosphorylation signal in the p.A617V variant compared with WT (n = 3). (b) Analysis of the temporal activation profile of MuSK over a 24‐hr period following incubation with full‐length human agrin. The pTyr/MuSK ratio from the different timepoints was calculated for each individual experiment relative to its maximum phosphorylation ratio (100%). The results are shown as mean ± SEM from n = 5 experiments. Repeated measures two‐way ANOVA (p = .003). Representative images of western blots are provided. IB, immunobinding; IP, immunoprecipitation; MuSK, muscle‐specific receptor tyrosine kinase; SEM, standard error of mean; WT, wild‐type

Figure 8

Molecular modeling of MuSK KD dimerization based on the EGFR‐KD dimer structure. (a) MuSK KD asymmetric dimer built by homology modeling based on the crystal structure of the EGFR‐KD activated dimer (MMDB ID: 40024). (b) Surface representation of MuSK KD dimer (green and cyan) based on the EGFR dimer. In the proposed structure, MuSK KDs, colored green and cyan, form an asymmetric dimer with the H‐helix (pink) of one monomer binding to αC‐helix (gray) and β4 sheet (orange) from the contralateral monomer. The p.Ala617 residue (red) is located within the αC‐helix at the dimerization interface. The tyrosines within the activation loop (Tyr‐750/754/755) are in blue. (c) The figure shows how the p.Ala617Val substitution modifies the dimerization interface increasing the hydrophobic interface for the docking of the contralateral MuSK KD. In this model, the p.Ala617 residue (red) interacts with the p.Ala842 and p.Asp843 residues (light purple) from the H‐helix (purple) at the dimerization interface. EGFR, epidermal growth factor receptor; KD, kinase domain; MuSK, muscle‐specific receptor tyrosine kinase