MuSK Glycosylation Restrains MuSK Activation and Acetylcholine Receptor Clustering

Watty, Anke; Burden, Steven J.

doi:10.1074/jbc.m208664200

Cited by 27 publications

(21 citation statements)

References 42 publications

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“…A mutation in the N-glycan consensus sequence of Musk, a muscle-specific receptor tyrosine kinase, results in an increase in ligand-independent activation (35). Thus, it is assumed that some N-glycans might act to prevent unnecessary proteinprotein interactions.…”

Section: Discussionmentioning

confidence: 99%

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

et al. 2007

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ErbB2 and ErbB3, two members of the ErbB family, form a high-affinity heregulin coreceptor that elicits potent mitogenic and transforming signals, and clinical studies indicate that these receptors play an important role in tumor incidence and progression. To determine whether N-glycosylation is involved in the function of ErbB3, a series of human ErbB3 molecules devoid of N-glycans were prepared and transfected to FlpIn-CHO cells for stable expression. A cross-linking study showed that the Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin. The wild-type or N418Q mutant of ErbB3 was next coexpressed with ErbB2 in Flp-In-CHO cells, and the effect of N-glycan on heterodimerization was examined. The N418Q mutant of ErbB3 was autodimerized with ErbB2 without ligand stimulation, and receptor tyrosine phosphorylation and subsequent extracellular signal-regulated kinase (ERK) and Akt phosphorylation were promoted in the absence of heregulin. A cell proliferation assay and a soft agar colony formation assay showed that the N418Q mutant of ErbB3 coexpressed with ErbB2 promoted cell proliferation and colony formation in soft agar in an ERK-and Akt-dependent manner. The mutation also promoted the growth of tumors in athymic mice when injected s.c. These findings suggest that the Asn 418 -linked N-glycan in ErbB3 plays an essential role in regulating receptor heterodimerization with ErbB2 and might have an effect on transforming activity. [Cancer Res 2007;67(5):1935-42]

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Section: Discussionmentioning

confidence: 99%

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

et al. 2007

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“…Modulation of agrin and MuSK activity by glycosylation is likely of great physiological relevance because they, along with rapsyn and AChRs, are the core components necessary for AChR cluster formation in vivo (35). Existing evidence suggests that neuraminidase induces AChR clustering in myotubes at least in part by removing sialic acid residues from oligosaccharides present on MuSK (31,36). Although peanut agglutinin could conceivably inhibit neuraminidaseinduced AChR clustering by directly binding to MuSK, Watty and Burden (36) showed that the sialic acid moieties present on MuSK were entirely contained within N-linked oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

“…Existing evidence suggests that neuraminidase induces AChR clustering in myotubes at least in part by removing sialic acid residues from oligosaccharides present on MuSK (31,36). Although peanut agglutinin could conceivably inhibit neuraminidaseinduced AChR clustering by directly binding to MuSK, Watty and Burden (36) showed that the sialic acid moieties present on MuSK were entirely contained within N-linked oligosaccharides. Moreover, this study observed no binding of peanut agglutinin to MuSK either before or after treatment with neuraminidase (36).…”

Section: Discussionmentioning

confidence: 99%

Core 1 Glycans on α-Dystroglycan Mediate Laminin-induced Acetylcholine Receptor Clustering but Not Laminin Binding

McDearmon

Combs

Ervasti

2003

Journal of Biological Chemistry

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Although unique O-linked oligosaccharides on ␣-dystroglycan are important for binding to a variety of extracellular ligands, the function(s) of more generic carbohydrate structures on ␣-dystroglycan remain unclear. Recent studies suggest a role for glycoconjugates bearing the core 1 disaccharide Gal␤(1-3)GalNAc in acetylcholine receptor (AChR) clustering on the surface of muscle cells. Here, we report experiments demonstrating that the core 1-specific lectin jacalin almost completely abrogated laminin-induced AChR clustering in C2C12 myotubes and that ␣-dystroglycan was the predominant jacalin-binding protein detected in C2C12 myotube lysates. Although jacalin likely inhibited laminin-induced AChR clustering by directly binding to ␣-dystroglycan, jacalin had no effect on laminin binding to the myotube surface or to ␣-dystroglycan. Like jacalin, peanut agglutinin lectin also binds the core 1 disaccharide but not when it is terminally sialylated as expressed on ␣-dystroglycan. We show that C2C12 ␣-dystroglycan bound to peanut agglutinin only after digestion with neuraminidase. Simultaneous treatment of myotubes with neuraminidase and endo-O-glycosidase diminished ␣-dystroglycan binding to peanut agglutinin and inhibited neuraminidase-induced AChR clustering. We conclude that sialylated core 1 oligosaccharides of ␣-dystroglycan are important for laminin-induced AChR clustering and that their function in this process is distinct from the established role of ␣-dystroglycan oligosaccharides in laminin binding.

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“…MuSK, the critical component of the receptor that transduces agrin's AChR aggregating activity, appears to be sialylated itself since neuraminidase treatment decreases the apparent size of the kinase on immunoblot gels (36) and since lectins specific to sialic acid bind to glycosylated MuSK (33). Sialic acid is thought to be attached to MuSK through two conserved N-linked glycosylation sites, one of which is on the second of three extracellular immunoglobulin domains that are involved in agrin binding.…”

Section: Possible Roles For Sialic Acid In Interactions Between Agrinmentioning

confidence: 99%

Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain

2007

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Agrin is a key heparan sulfate proteoglycan involved in the development and maintenance of synaptic junctions between nerves and muscles. Agrin's important functions include clustering acetylcholine receptors on the postsynaptic membranes of muscles and binding to the muscle protein α-dystroglycan through its glycan chains. ITC and NMR were used to study the interactions of the Cterminal domain, agrin-G3, with carbohydrates implicated in agrin's functions. Sialic acid caps the glycan chains of α-dystroglycan and occurs as a posttranslational modification on the muscle-specific kinase component of the agrin receptor. We found that agrin-G3 binds sialic acid in a Ca 2+ -dependent manner. ITC data indicate that binding is exothermic and occurs with a 1:1 stoichiometry. NMR chemical shift changes map the sialic acid binding site to the loops that control the domain's acetylcholine receptor clustering activity. By contrast, the glycosaminoglycans heparin and heparan sulfate bind independently of Ca 2+ . Binding is endothermic, and the binding site spans about 12 saccharide units. The binding site for heparin occupies a similar location but is distinct from that for sialic acid. NMR translational diffusion experiments show that agrin-G3 binds heparin with a 2:1 stoichiometry. Comparisons between the muscle (B0) and neuronal (B8) isoforms of the agrin domain showed very similar Ca 2+ and carbohydrate binding properties. Our work identifies agrin-G3 as a functional analogue of the concanavalin A-type lectins, highlights functional similarities between agrin and laminin G domains, and provides mechanistic clues about the roles of carbohydrates in agrin's functions.The heparan sulfate proteoglycan agrin is a critical component of neuromuscular junctions (NMJs) 1 (1). It has additional less well understood roles in the central nervous system (2,3), nonneural tissues (4), immunological synapses (5), and amyloid diseases (6,7). The agrin core protein consists of 22 domains. At the C-terminus are three globular G-domains separated by epidermal growth factor-like domains. The G-domains have a β-jellyroll folding motif structure (8) that belongs to the LNS superfamily, which includes domains from laminin, neurexin, and steroid-hormone binding globule (9). Beyond the LNS family, the closest structural relatives are the pentraxins: serum amyloid P component and C-reactive protein. 1 Abbreviations: ACh, acetylcholine; AChR, acetylcholine receptor; α-DG, α-dystroglycan; G-domain, globular domain; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation; ITC, isothermal titration calorimetry; LG, laminin globular domain; LNS, laminin, neurexin, and steroid-hormone binding globule; MASC, myotube-associated specificity component; MuSK, muscle-specific kinase; NCAM, neural cell adhesion molecule; NMJ, neuromuscular junction; NMR, nuclear magnetic resonance; PFG-LED, pulse field gradient longitudinal encode-decode. One function of agrin is to form a structural bridge between the NMJ basal lamina and muscle ...

show abstract

MuSK Glycosylation Restrains MuSK Activation and Acetylcholine Receptor Clustering

Cited by 27 publications

References 42 publications

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

The Asn418-Linked N-Glycan of ErbB3 Plays a Crucial Role in Preventing Spontaneous Heterodimerization and Tumor Promotion

Core 1 Glycans on α-Dystroglycan Mediate Laminin-induced Acetylcholine Receptor Clustering but Not Laminin Binding

Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain

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