MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction - PubMed (original) (raw)

MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction

L Strochlic et al. J Cell Biol. 2001.

Abstract

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.

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Figures

Figure 1

Figure 1

MuSK cross-link products and immunopurification of MuSK complexes. (a) Proteins from Torpedo AChR-rich membranes were cross-linked with various concentrations of SMPB (lane 0, control; lane 1, 7 × 10−6 M; lane 2, 2 × 10−5 M; lane 3, 4 × 10−5 M; lane 4, 10−4 M; lane 5, 2.5 × 10−4 M) and separated by SDS-PAGE. Western blots revealed that in addition to MuSK (97 kD), a 140-kD cross-link product was detected at low SMPB concentrations. Other cross-link products were observed at higher concentrations. (b) Purification of uncross-linked (−SMPB) and cross-linked (+SMPB, 4 × 10−5 M) MuSK complexes was achieved by immunoaffinity chromatography. The immunopurified polypeptides or the MuSK cross-link products were detected by silver staining (ST) or identified by immunoblotting (IB), respectively. In both experiments, a 40-kD polypeptide was detected in addition to MuSK. After SMPB treatment, a major cross-link product of 140 kD was detected. The asterisk indicates residual IgGs.

Figure 2

Figure 2

MALDI-TOF mass spectometry analysis of Torpedo MuSK binding partners. (a–c) Coverage maps of experimental tryptic peptides compared with theoretical tryptic peptides from databases obtained with ProFound™ software were shown. Coverages of 6% with rat MuSK (a), and of 16% with the COOH-terminal peptide (residues 1124–1374) of mouse MAGI-1c (b) were obtained from the 140-kD cross-link product. A coverage of 35% with the COOH-terminal peptide of MAGI-1c was obtained from the 40-kD polypeptide coimmunopurified with MuSK (c).

Figure 3

Figure 3

Characterization of the MuSK–MAGI-1c interaction in Torpedo postsynaptic membranes. (a) Western blots of AChR-rich Torpedo membranes were probed with preimmune serum (lane 1), and anti–MAGI-1c (R 85) (lane 2). A bona fide MAGI-1c (150 kD) and a 40-kD putative proteolytic fragment were detected (lane 2). (b) Western blots probed with R 497 and mab 2847 antibodies show that full-length MAGI-1 and MuSK were present together in a 250-kD cross-link product. (c) MuSK was coimmunopurified with the COOH-terminal fragment of MAGI-1c upon immunoaffinity chromatography with R 84 or R 85 antibodies. The immunopurified polypeptides were detected by silver staining (ST), and MuSK was detected by immunoblotting.

Figure 4

Figure 4

MAGI-1c localizes at the Torpedo and rat cholinergic synapses. (a) Double-fluorescence experiment showing the colocalisation of MAGI-1 (antibody R 499) and AChRs (α-bungarotoxin staining) at the innervated faces of Torpedo electrocytes. (b) In rat skeletal muscle fibers, MAGI-1 (antibody R 499) also strictly colocalized with AChRs at the NMJs. (c) At variance, MAGI-1 was not detected in agrin-induced AChR clusters in C2C12 myotubes. Bars, 30 μm.

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