Journal of Cell Science 113, 2877-2886 (2000) 2877 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1377 Laminin and α7β1 integrin regulate agrin-induced clustering of acetylcholine receptors Dean J. Burkin, Jae Eun Kim, Maojian Gu and Stephen J. Kaufman Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA *Author for correspondence (e-mail: [email protected]) Accepted 1 June; published on WWW 20 July 2000 SUMMARY The clustering of acetylcholine receptors (AChRs) in the is independent of clustering. In addition to laminin-1, post-synaptic membrane of skeletal muscle is an early merosin (laminin-2/4) is present both before and after developmental event in the formation of the neuromuscular formation of neuromuscular junctions and also promotes junction. Several studies show that laminin, as well as AChR clustering and colocalization with the integrin neural agrin, can induce AChR clustering in C2C12 as well as synergism with agrin. Using site directed myofibers. We recently showed that specific isoforms of mutagenesis we demonstrate that a tyrosine residue in the the α7β1 integrin (a receptor normally found at cytoplasmic domain of both α7A and α7B chains regulates neuromuscular junctions) colocalize and physically the localization of the integrin with AChR clusters. We also interact with AChR clusters in a laminin-dependent provide evidence that laminin, through its association with fashion. In contrast, induction with agrin alone fails to the α7β1 integrin, reduces by 20-fold the concentration of promote localization of the integrin with AChR clusters. agrin required to promote AChR clustering and accelerates Together both agrin and laminin enhance the interaction of the formation of clusters. Thus laminin, agrin and the the integrin with AChRs and their aggregation into α7β1 integrin act in a concerted manner early in the clusters. To further understand this mechanism we development of the post-synaptic membrane, with laminin investigated cluster formation and the association of the priming newly formed myofibers to rapidly and vigorously α7β1 integrin and AChR over time following induction respond to low concentrations of neural agrin produced by with laminin and/or agrin. Our results show that the α7β1 innervating motor neurons. integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable Key words: α7β1 integrin, Laminin, Agrin, Acetylcholine receptor, association of the integrin and AChRs and this association Neuromuscular junction INTRODUCTION Neural agrin is an extracellular matrix protein derived from the innervating motor neuron. Whereas AChRs are diffusely The post-synaptic membrane of the neuromuscular junction dispersed on newly developed, uninnervated skeletal muscles, (NMJ) develops from the coordinated localization of proteins agrin initiates clustering of AChRs in vitro and in vivo (Fertuck to the sites where motor neurons innervate muscle. Although and Salpeter, 1976; Frank and Fishbach, 1979; McMahan, many of the proteins that comprise the NMJ have been defined, 1990; Bowe and Fallon, 1995; Anderson and Cohen, 1997; the process by which these proteins are recruited to this site Reugg and Bixby, 1998). This aggregation is believed to result remains unclear. What is certain is that communication from the interaction of agrin with a receptor complex on the mediated by nerve and muscle derived extracellular matrix membrane of myofibers that includes MuSK (a muscle specific proteins and cell surface receptors is central to this process. tyrosine kinase; Valenzuela et al., 1995; DeChiara et al., 1996; These interactions trigger the mobilization of proteins Glass et al., 1996). The αvβ1 integrin (Martin and Sanes, 1997) including acetylcholine receptors (AChRs), rapsyn, muscle and α-dystroglycan (Sugiyama et al., 1994; Campanelli et al., specific kinase (MuSK), α7β1 integrin, α and β dystroglycans, 1994) have also been suggested to function as co-receptors for sacroglycans, utrophin, erbB and syntrophins to sites of motor agrin. The interaction of agrin with its proposed receptor neuron innervation and initiate synapse-specific transcription complex leads to the phosphorylation of MuSK and the β- of genes encoding many neuromuscular proteins. The subunit of the AChR (Glass et al., 1996). Induction of interaction of these and other proteins in the post-synaptic clustering by agrin promotes a stronger physical interaction membrane leads to the development, stabilization and between AChRs and MuSK (Furher et al., 1999). maintenance of the adult neuromuscular synapse. For a recent Recent in vitro studies report that laminin-1 (LN-1), a review, see Sanes and Lichtman (1999). component of the developing muscle extracellular matrix 2878 D. J. Burkin and others (Patton et al., 1997), can also initiate AChR clustering (Gibco BRL, Gaithersberg, MD), 2 mM glutamine, 100 units/ml independently of agrin and MuSK (Vogel et al., 1983; penicillin, 100 µg/ml streptomycin and 10 µg/ml kanamycin. C2C12 Sugiyama et al., 1997; Montanaro et al., 1998). Furthermore, cells transfected to express the rat α7AX2 and α7BX2 integrin chains upon induction of clustering with laminin, the β-subunit of the under control of the mouse muscle creatine kinase promoter have been previously described (Burkin et al., 1998). The α7B chain cytoplasmic AChR is not phosphorylated by MuSK (Sugiyama et al., 1997). α These results led to the suggestion that agrin and laminin domain mutants 7BX2-YTF (tyrosine to phenylalanine mutation), α7BX2-DXHP (deletion of the DXHP repeat domain) and α7AX2- initiate distinct pathways for AChR clustering (Sugiyama et al., YTF (tyrosine to phenylalanine mutation) were constructed using site 1997). However, in the presence of both agrin and laminin, directed mutagenesis (Stratagene) following the manufacturer’s there is an enhanced aggregation response: larger AChR protocol. These α7 chain mutations were subcloned into the pBKRSV clusters form faster, and in greater numbers (Sugiyama et al., vector (Stratagene) containing the MCK promoter (Jaynes et al., 1997; Burkin et al., 1998). 1986) using the restriction enzyme sites BstEII and KpnI. All The α7β1 integrin is a laminin receptor that serves as a constructs were verified by DNA sequencing. Purified plasmids were transmembrane link and signal transduction mechanism digested with EcoRI to linearize the construct and transfected into between the extracellular matrix and muscle fiber (Song et al., C2C12 mouse myoblast cells. Stable cell populations were selected 1992; Hodges and Kaufman, 1996; Burkin and Kaufman, in growth media containing G418 as described (Burkin et al., 1998). 1999). Alternative cytoplasmic domains (A, B and C; Song et Immunoprecipitation and immunoblot analysis al., 1993; Collo et al., 1993; Ziober et al., 1993) and Approximately 2×105 C2C12 myoblasts transfected with the MCK- extracellular domains (X1 and X2; Ziober et al., 1993; Hodges rat α7BX2 construct were seeded on fibronectin coated (20 µg/ml), and Kaufman, 1996) are generated by developmentally 100 mm diameter tissue culture dishes. At confluence, differentiation regulated alternative RNA splicing. In the adult, different medium (2% horse serum, no embryo extract) was added to induce isoforms of this integrin are concentrated at the neuromuscular myotube formation. AChR clustering was induced with 60 nM and myotendinous junctions, as well as extrajunctionally laminin-1, 0.5 nM agrin, or 60 nM laminin-1 and 0.5 nM agrin for 0, (Martin et al., 1996; Hodges and Kaufman, 1996; Burkin and 2, 4, 6, 8 or 16 hours. Cells were washed once in ice-cold phosphate Kaufman, 1999). This integrin also has a functional role in the buffered saline (PBS) (without Ca2+ or Mg2+) and harvested in ice- cold PBS containing 2 mM PMSF. Cells were extracted at 4°C in 200 development of the postsynaptic membrane. Specific spliced β isoforms of α7β1 integrin (α7AX2 and α7BX2) participate in mM octyl- -D-glucopyranoside, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 2 mM PMSF, 20 µg/ml aprotinin, and 12.5 µg/ml laminin-induced AChR clustering (Burkin et al., 1998). Upon leupeptin. Extracts were incubated at 4oC with anti-AChR antibody induction of clustering with laminin, or laminin and agrin, all K20 (Santa Cruz Biotechnology, Santa Cruz, CA) at 10 µg AChRs colocalize with the integrin. In contrast, little antibody/mg total protein for 12 hours. Protein G agarose (Sigma, St colocalization is seen upon agrin-induced clustering. High Louis, MO) was added at 1 mg Protein G/100 mm plate of cells, for concentrations of anti-α7 antibodies inhibit colocalization of 4 hours at 4°C. The beads were then centrifuged at 4°C, at 12,000 the integrin and AChR as well as the enhanced response rpm and washed three times in extraction buffer. The beads were then promoted by both laminin and agrin. These results suggest that mixed with 2× sample buffer, heated to 65°C for 5 minutes, and agrin, laminin and the α7β1 integrin interact in concert in the centrifuged at 14,000 rpm, at 4°C, for 5 minutes. The supernatants formation of the neuromuscular junction. were collected and the proteins were separated on 8% polyacrylamide To further understand the localization and role of the α7β1 SDS gels, at 40 mA, for 50 minutes. The proteins were transferred to nitrocellulose filters. Blocked filters were incubated with a 1:500 integrin in the development of the neuromuscular junction, we dilution of polyclonal