INTRODUCTION A key event in the formation of synapses is the accumulation of neurotransmitter receptor proteins at the postsynaptic membrane, directly opposite the presynaptic nerve terminal. During the development of the neuromuscular junction (NMJ), the nicotinic acetylcholine receptor (AChR) undergoes a dramatic relocalisation and aggregates at high density in the plasma membrane juxtaposed to the nerve terminal (Hall and Sanes, 1993). The innervating motor nerve regulates this accumulation through synapse-specific induction of gene expression and via post-translational mechanisms (Duclert and Changeux, 1995). The extracellular matrix (ECM) protein agrin is involved in the post-translation clustering of AChRs and also plays a role in the induction of synapse-specific gene expression (reviewed by Ruegg and Bixby, 1998). Members of the dystrophin-associated protein complex (DPC) and dystrophin-related proteins have been implicated in synaptic structure and function. The dystrophin-deficient mdx mouse has an increase in the rate of AChR degradation similar to that observed following denervation (Xu and Salpeter, 1997). These mice also have morphologically abnormal NMJs, characterised by a reduction in the number of junctional folds (Lyons and Slater, 1991). The dystrophin-associated protein, α-dystroglycan, is the major agrin-binding protein in muscle and other tissues; however, α-dystroglycan is unlikely to be a functional agrin-receptor (Meier et al., 1996; Gesemann et al., 1998). Although the absence of dystrophin results in loss of the DPC from the extra-synaptic sarcolemma, dystrophin- associated proteins are still retained at the NMJ through an association with the dystrophin-related protein, utrophin (Matsumara et al., 1992). In normal adult muscle utrophin is found exclusively at the NMJ (Ohlendieck et al., 1991). Similar to the mdx mouse, utrophin-deficient mice have a reduction in the number of junctional folds at a single synapse (Deconinck et al., 1997; Grady et al., 1997). Thus, the specialised postsynaptic cytoskeleton of the NMJ appears to contain a modified DPC that is required to maintain normal synaptic structure. To determine the function of other dystrophin-related proteins at the synapse, we have focused our studies on α- dystrobrevin (also known as 87K protein or A0). Dystrobrevin was originally identified in the Torpedo electric organ as a component of the postsynaptic membrane that co-purified with 2595 Journal of Cell Science 111, 2595-2605 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS4587 Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of α-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce α- dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct α-dystrobrevin isoforms. In myotubes, α- dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, α-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of α-dystrobrevin-1 by agrin. Furthermore, we show that α-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus α-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that α- dystrobrevin-1 is not involved in the phosphorylation- dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin. Key words: Dystrobrevin, Utrophin, Agrin, Acetylcholine receptor (AChR) clustering, Muscle SUMMARY Characterisation of α-dystrobrevin in muscle Ralph Nawrotzki 1, *, Nellie Y. Loh 1 , Markus A. Ruegg 2 , Kay E. Davies 1,‡,§ and Derek J. Blake 1 1 Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK 2 Department of Pharmacology, Biozentrum, University of Basle, CH-4056 Basle, Switzerland *Present address: Max-Planck-Institut fuer Biochemie, Abteilung Proteinchemie, D-82152 Martinsried bei Muenchen, Germany ‡ Present address: Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK (e-mail: kdavies@bioch.ox.ac.uk) § Author for correspondence Accepted 1 July; published on WWW 13 August 1998