At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

The accumulation of dystrophin and associated proteins at the postsynaptic membrane of the neuromuscular junction and their co-distribution with nicotinic acetylcholine receptor (AChR) clusters in vitro suggested a role for the dystrophin complex in synaptogenesis. Co-transfection experiments in which ␣-and ␤-dystroglycan form a complex with AChR and rapsyn, a peripheral protein required for AChR clustering (Apel, D. A., Roberds, S. L., Campbell, K. P., and Merlie, J. P. (1995) Neuron 15, 115-126), suggested that rapsyn functions as a link between AChR and the dystrophin complex. We have investigated the interaction between rapsyn and ␤-dystroglycan in Torpedo AChR-rich membranes using in situ and in vitro approaches. Cross-linking experiments were carried out to study the topography of postsynaptic membrane polypeptides. A cross-linked product of 90 kDa was labeled by antibodies to rapsyn and ␤-dystroglycan; this demonstrates that these polypeptides are in close proximity to one another. Affinity chromatography experiments and ligand blot assays using rapsyn solubilized from Torpedo AChR-rich membranes and constructs containing ␤-dystroglycan C-terminal fragments show that a rapsyn-binding site is present in the juxtamembranous region of the cytoplasmic tail of ␤-dystroglycan. These data point out that rapsyn and dystroglycan interact in the postsynaptic membrane and thus reinforce the notion that dystroglycan could be involved in synaptogenesis.