Journal of Cell Science 113, 2877-2886 (2000) 2877 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1377

Laminin and α7β1 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 -1, post-synaptic membrane of 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 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 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 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 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 rabbit anti-α7CDB (347) antisera that have investigated the recruitment of the integrin to AChR recognizes the α7B cytoplasmic domain (Song et al., 1993). clusters over time, after induction with laminin and/or agrin. Immunoreactive proteins were detected using HRP-conjugated Site directed mutagenesis of specific amino acids in the α7 donkey anti-rabbit immunoglobulin (Amersham ECL kit) following cytoplasmic domain allowed an investigation of the role of the manufacturer’s directions. these residues in the localization of the integrin with AChR clusters. Our results show the α7β1 integrin is recruited early AChR cluster analysis and immunofluorescence during the induction of AChR clustering, that laminin LAB-TEK eight-well chamber slides (No. 177445; Nalge Nunc) were µ modulates this recruitment and that a tyrosine residue in the coated with purified human fibronectin (20 g/ml) for 24 hours and cytoplasmic domain of both α7A and α7B integrin isoforms washed with PBS before cells were seeded. The plates were seeded with 1.5×104 myoblasts in 0.15 ml growth medium per well, and regulates the association of the integrin with AChRs. incubated 3 days at 37°C, in 5% CO2, until confluent. Differentiation Moreover, we provide evidence that laminin, through its medium containing 2% horse serum and 2 mM glutamine was then association with the α7β1 integrin, reduces the threshold added to induce myotube formation (Burkin et al., 1998). After 3 days, concentration of agrin required to induce the aggregation of mature myotubes were treated with 60 nM laminin-1 (Gibco-BRL) AChRs. Thus, laminin and agrin work in a concerted manner and/or 0.5 nM recombinant COOH-terminal (N2) agrin for 2, 4, 6, 8 early in the formation of the neuromuscular junction with or 16 hours. Human placental merosin, (laminin-2/4) (Gibco-BRL) laminin priming newly formed myofibers to rapidly respond to was dialyzed for two days against Dulbecco’s medium; 90 nM was extremely low concentrations of agrin. used to induce clustering. Identical results were obtained using human placental merosin prepared as described (Ehrig et al., 1990). Agrin was prepared from Chinese hamster ovary (CHO) cells stably transfected to secrete N2 neural agrin into the growth medium. These MATERIALS AND METHODS cells were grown to confluence and serum deficient medium was added for 24-36 hours. Medium containing agrin was collected and Cell lines centrifuged to remove cell debris. The concentration of agrin was C2C12 mouse myoblasts were cultured in Dulbecco’s medium (low determined by western analysis using the anti-agrin monoclonal glucose) containing 20% fetal calf serum, 0.5% chick embryo extract antibody (Mab 540). Band intensities were quantitated (using NIH Laminin and α7β1 integrin mediated AChR clustering 2879 image software) and compared with bands obtained with known compared to either inducer alone; the maximum number of concentrations of agrin. Activity of agrin was determined by AChR clusters were produced 4 hours after induction. With all comparing the number of AChR clusters induced in C2C12 myotubes inducers and cell lines, towards the end of the 16 hour time with the number of clusters induced using agrin of known course the number of AChR clusters declined. These results concentrations. concur with previous observations that clusters that first form For preincubation experiments, laminin was added for two hours, subsequently fuse to form larger aggregates (Olek et al., 1986; the medium was then removed, the cells were washed with warm Kong and Anderson, 1999). medium, and various concentrations of agrin in differentiation α medium were added as indicated for an additional 4 hours. Fig. 2B and D show the colocalization of the 7AX2 and Myotubes were stained for 1 hour at 37°C with O26 mouse α7BX2 integrin isoforms with AChR clusters over time. monoclonal antibody (10 µg/ml) to detect the rat α7 integrin (Song Within 2 hours of induction with 60 nM laminin or 0.5 nM et al., 1992) and with rhodamine labeled α-bungarotoxin (Molecular agrin and 60 nM laminin, 75-85% of AChR clusters Probes; 1:1000 dilution) to detect AChR clusters. Myotubes were then colocalized with the α7AX2 and α7BX2 integrin isoforms. washed in Dulbecco’s modified Eagle’s medium and stained with This level of colocalization is maintained throughout the time FITC-labeled goat anti-mouse antibody (Jackson Immunoresearch) course. Upon induction with agrin alone, low levels of for 45 minutes to visualize the O26 antibody. The cells were then colocalization between AChR clusters and the α7AX2 and washed and fixed in 100% ice-cold methanol for 5 minutes, hydrated α7BX2 are detected (5-25%). Induction with both in PBS, and mounted in PBS:glycerol containing 10 µM p- phenylenediamine. All samples were done in triplicate. AChR clusters laminin and agrin enhances this response. Thus laminin and α7 localization were visualized using a Zeiss Photomicroscope induces the rapid colocalization of integrin with AChR clusters III and ×63 Planapo objective. Images of immunofluorescence and this high level of colocalization persists. localization were acquired using a Sony DXC9000 color video CCD camera. The α7β1 integrin physically associates with AChRs in the presence of laminin Previous results showed the α7β1 integrin is physically RESULTS associated with AChRs in the presence of laminin 16 hours after induction (Burkin et al., 1998). Because the In earlier experiments we demonstrated that laminin, through immunofluorescence data showed that the integrin rapidly co- its interaction with specific alternative cytoplasmic domain localized with AChR clusters, we determined if the integrin isoforms of the α7β1 integrin (α7X2B and α7X2A), promotes and AChRs were also physically associated early in cluster the physical association of the integrin and AChR as well as formation. C2C12-α7BX2 myotubes were induced with agrin, co-clustering of these two receptors (Burkin et al., 1998). laminin, or both agrin and laminin, and myotubes were Induction of AChR clustering by both agrin and laminin harvested at 0, 2, 4, 6, 8, and 16 hours after induction. Anti- produces a synergistic aggregation and all AChRs colocalize AChR antibody and Protein-G beads were used to with the integrin (Burkin et al., 1998). In contrast, AChR immunoprecipitate AChRs. Western analysis, using anti-α7 aggregates induced by agrin only minimally co-cluster and do antisera CDB2, was used to detect the integrin. As seen in Fig. not physically associate with the integrin (Fig. 1). As before, 3, the α7 integrin chain (120 kDa band and 68 kDa fragment) we have used mouse C2C12 myogenic cells, transfected to are co-precipitated with AChRs and are first detectable in express the rat α7 integrin, and anti-rat α7 antibodies to precipitates 2 hours after induction with either 60 nM laminin localize the ectopically expressed α7 integrin chain. or 60 nM laminin and 0.5 nM agrin. This association between α7 integrin and AChR is maintained throughout the duration The α7β1 integrin is recruited early during AChR of the experiment, with maximum association detected 4 hours clustering after induction. To determine when the α7β1 integrin is recruited with AChR The association of the integrin with AChR at 4 hours and clusters, co-clustering of the α7β1 integrin with AChRs thereafter is equivalent following induction with either laminin, induced by 0.5 nM agrin, 60 nM laminin, or 60 nM laminin or laminin and agrin (Fig. 3), whereas both inducers are and 0.5 nM agrin was determined over a period of 16 hours. required for enhanced clustering (Fig. 2A and C). This suggests Only AChR clusters with greater than 50% coincident α7 chain that in the absence of agrin and extensive clustering, laminin and AChR signals were scored as colocalized. Three cell alone is sufficient to promote the stable physical association of populations were used: α7AX2 (C2C12 cells transfected to the α7β1 integrin and AChR. express the rat α7AX2 isoform), α7BX2 (C2C12 cells In contrast, the α7 integrin is not immunoprecipitated with transfected to express the rat α7BX2 isoform), and AChRs upon induction with agrin alone. Upon prolonged untransfected C2C12 cells. exposure, a basal level of association between the integrin and Fig. 2A,C,E show the numbers of AChR clusters formed 0, AChR is detected in the absence of added inducers; this may 2, 4, 6, 8, 12 and 16 hours after induction of α7AX2, α7BX2 reflect the formation of ‘spontaneous’ AChR clusters promoted and C2C12 myotubes, respectively. In all cell lines, the by nascent laminin. After induction with agrin, less α7 chain numbers of AChR clusters induced with 60 nM laminin is co-precipitated with AChR. This suggests that in the absence increased slowly and attained a maximum 12-16 hours after of laminin, agrin may promote the dissociation of the integrin induction. Receptor clustering induced with 0.5 nM agrin was and AChR. somewhat faster compared to laminin, with maximum numbers reached 4-6 hours after induction. Most notably, in the Preincubation with laminin enhances AChR cluster presence of both 0.5 nM agrin and 60 nM laminin, AChR formation cluster formation was both more rapid and more extensive Prior to innervation by motor neurons muscle fibers in situ are 2880 D. J. Burkin and others surrounded by an extracellular matrix that is rich in laminin AChR clustering (Burkin et al., 1998) and are present at the (Bayne et al., 1984; Patton et al., 1997). This suggested that neuromuscular junction in adult skeletal muscle (Martin et al., laminin may prime fibers to react synergistically with agrin 1996). To investigate the role of the α7 integrin cytoplasmic during development of the neuromuscular junction. To domain in regulating the recruitment of the integrin to AChR examine this, we determined whether laminin can prime AChR clusters, we studied the localization of three mutant α7 chains clustering in vitro and thereby render myofibers receptive to to these clusters. All isoforms of the α7 integrin have a single subsequent induction by neural agrin. C2C12-α7BX2 tyrosine residue in their cytoplasmic domains. In the α7B myotubes were preincubated for 2 hours with 60 nM laminin. isoform, this tyrosine lies within a phosphotransfer motif The unbound laminin was then removed, and 0.5 nM agrin was common to serine/threonine kinases and a region that is 80% then added for a further 2 hours. homologous with a hydrophobic sequence in the family of Preincubation of myotubes with laminin for 2 hours, receptor-like protein tyrosine phosphatases (Song et al., 1993). followed by addition of agrin for 2 hours, resulted in a 2.4-fold To date there has been no evidence to show that this tyrosine increase in the number of AChR clusters compared to residue is involved in regulating any biological process. To myotubes simultaneously induced with agrin and laminin for investigate the possible role of this tyrosine residue in AChR 2 hours (Fig. 4). The number of AChR clusters produced by clustering, site directed mutagenesis was used to change this laminin alone over 4 hours was low, and the number of AChR tyrosine in both rat α7A and α7B isoforms to phenylalanine clusters produced solely by agrin over 2 hours could also not (YTF). These altered constructs were cloned so that their account for the large increase in clusters seen after expression was regulated by the mouse muscle creatine kinase preincubation with laminin. This suggests that laminin can promoter. This resulted in the altered integrin constructs prime myofibers to vigorously respond to agrin. α7AX2-YTF and α7BX2-YTF. An additional mutation in the Preincubation with laminin lowers the concentration of agrin required for AChR clustering To determine the physiologic rationale for a dual induction system, we asked whether priming muscle fibers with laminin alters the threshold concentration of agrin required to induce AChR aggregation. C2C12-α7BX2 myofibers were preincubated with laminin for 2 hours. Unbound laminin was then removed, and various concentrations of agrin (0.1-500 pM) were added for an additional 4 hours. Preincubation with laminin resulted in a 20-fold reduction in the concentration of agrin required to initiate clustering (Fig. 5). Preincubation with laminin accelerates AChR cluster formation Preincubation with laminin was also found to accelerate cluster formation. Myofibers were primed with laminin for 2 hours, as above, and then induced with 0.2 nM agrin for 5 minutes to 6 hours. Cluster formation in primed fibers was compared to clusters formed upon simultaneous addition of laminin and agrin (Fig. 6A). Clusters are detected in primed fibers within 10 minutes. In contrast, 30-60 minutes are required in the absence of priming. The rates of cluster formation thereafter are identical (Fig. 6B). Thus preincubation with laminin both lowers the threshold concentration of agrin required to initiate AChR aggregation and accelerates the development of the neuromuscular junction.

A cytoplasmic tyrosine residue in the α α7 chain regulates integrin Fig. 1. Immunofluorescence localization of the rat 7 integrin (green) with AChR clusters (red). The wild-type α7BX2 chain expressed in transfected C2C12 cells localizes with colocalization with AChR clusters laminin and agrin induced AChR clusters but not with agrin induced AChR clusters. Both the α7A and α7B alternative Induction with both laminin and agrin promotes more vigorous clustering. Endogenous α7 cytoplasmic domain isoforms function in integrin in C2C12 myotubes does not react with the O26 anti-rat α7 antibody. Bar, 15 µm. Laminin and α7β1 integrin mediated AChR clustering 2881

AX2-Laminin BX2-Laminin A AX2-Agrin C BX2-Agrin 20 AX2-Agrin+Laminin BX2-Agrin+Laminin 20

15 15

10 10

5 AChR clusters/field

AChR clusters/field 5

0 0 2 4 6 8 10 12 14 16 0 0 2 4 6 8 10 12 14 16 Time (hours) Time (hours)

B AX2-Laminin BX2-Laminin AX2-Agrin D BX2-Agrin AX2-Agrin+Laminin 100 100 BX2-Agrin+Laminin

80 80

60 60

40 40 Percent Colocalization

Percent Colocalization 20 20

0 0 2 4 6 8 12 16 0 0 2 4 6 8 12 16

Time (hours) Time (hours)

C2C12-Laminin E C2C12-Agrin 20 C2C12-Agrin+Laminin Fig. 2. Induction and colocalization of AChR clusters with α7β1 integrin in C2C12 myofibers and fibers expressing the rat α7AX2 and α7BX2 isoforms. (A and C) The number of AChR clusters induced by 0.5 nM agrin increases and peaks at 2-4 hours. The number of 15 AChR clusters induced by 60 nM laminin-1 increases more slowly and peaks at 12-16 hours. The numbers of AChR clusters produced after induction with both 0.5 nM agrin and 60 nM laminin is much greater than produced by either inducer alone, with the highest 10 numbers observed 4 hours after induction. (B and D) Colocalization of the ectopic rat integrin with AChR clusters. In the presence of both agrin and laminin-1, α7AX2 and α7BX2 isoforms rapidly localize (2 hours) with 70-85% of AChR clusters. In contrast, agrin alone does AChR clusters/field 5 not promote co-localization of the integrin and AChRs at any point during the time course. (E) The number of AChR clusters scored in C2C12 myotubes after induction with agrin and/or laminin-1. AChR cluster numbers are similar between transfected and non-transfected 0 0 2 4 6 8 10 12 14 16 myotubes. The mean values scored in triplicate samples ± s.e., are given. Time (hours) 2882 D. J. Burkin and others

Fig. 3. Coprecipitation of the AChR and the α7β1 integrin. Anti-AChR antibody was used to immunoprecipitate AChR from extracts prepared from C2C12-α7BX2 myotubes at different times after induction with 0.5 nM agrin, 60 nM laminin-1 or both. Western blot analysis using anti-α7 cytoplasmic domain B antibody, detects the integrin in the complex. A stable physical interaction between AChRs and the α7β1 integrin is promoted by induction with laminin or laminin and agrin within 2-4 hours. Laminin alone promotes a physical interaction between the integrin and AChRs before AChR clusters are visible, thus clustering is not required for the association of the integrin and AChRs. This association is maintained throughout the time course. In contrast, induction with agrin does not promote a physical association between the integrin and AChR clusters at any point during the time course.

α7B cytoplasmic domain was also made: the threefold DXHP 50 repeat in the α7B cytoplasmic domain, that appears to be unique to this integrin alpha chain, was deleted. This mutant 40 construct was cloned as above and these mutant rat α7 cDNA constructs were transfected into mouse C2C12 myoblasts. Stable cell populations were isolated and identified by western 30 and immunofluorescence analyses of mature myofibers using antibodies specific to the rat α7 chain. 20 Cluster numbers and colocalization of the integrin with AChR clusters were determined in myofibers expressing the rat 10 α7 chain mutants, the respective wild-type integrins, and AChR clusters/field untransfected C2C12 myofibers (Fig. 7). No significant differences in AChR cluster numbers were found between any of 0 the cell lines. Furthermore, the α7X2-YTF mutants colocalized normally with AChRs upon induction with laminin or with None laminin and agrin compared to controls (80-95%). In contrast, Ag 2hrs LN 4hrs LN 6hrs whereas agrin-induced colocalization of wild-type α7AX2 and Ag+LN 2hrs α7BX2 with AChRs is minimal (5-15%), myofibers expressing the mutant α7AX2-YTF and α7BX2-YTF α7 chains showed high levels of colocalization (60-70%). This change in Inducer colocalization of the mutated integrin upon induction with agrin suggests that the α7 cytoplasmic domain tyrosine residue is a

negative regulator of agrin-induced localization of the integrin LN preincubation 2hrs+Ag 2hrs LN preincubation 4hrs+Ag 2hrs with AChRs. This regulation could be at the level of phosphorylation of the α7 chain, thereby changing the integrin’s Fig. 4. Preincubation with laminin primes myofibers for agrin- induced AChR clustering. C2C12 myofibers were preincubated interactions with other proteins, or due to conformational with 60 nM laminin-1 for 2 or 4 hours and unbound laminin was changes produced by substituting phenylalanine at these sites. then removed. Addition of 0.5 nM agrin for 2 hours to the Deletion of the three DXHP motifs in the α7BX2 preincubated myofibers resulted in 2-2.5 times more AChR clusters cytoplasmic domain had no apparent affect on the formation than obtained upon incubation with 0.5 nM agrin and 60 nM of clusters or on colocalization of the rat integrin and AChRs laminin together for 2 hours. The mean values scored in triplicate induced by agrin, laminin, or agrin and laminin (Fig. 7). samples ± s.e., are given. Laminin-2/4 induces acetylcholine receptor clustering and enhances agrin-induced cluster formation laminin-4 (α2β2γ1), laminin-9 (α4β2γ1), and laminin-11 Different isoforms of the αβγ laminin heterotrimer are (α5β2γ1) are localized at synapses (Patton et al., 1997). It was expressed at different times and sites before and subsequent to therefore of interest to determine whether isoforms other than the development of skeletal muscle (Patton et al., 1997). laminin-1 would also promote AChR clustering. Whereas Laminin-1 (α1β1γ1) is made by myoblasts and laminin-2 Sugiyama et al. (1997) reported that commercial merosin (a (α2β1γ1) is made by newly formed myotubes in vitro (Vachon mixture of laminin-2 and laminin-4; Vachon et al., 1997; et al., 1996). Laminin α4 and α5 chains are also expressed by Brown et al., 1994) did not promote AChR clustering, more myogenic cells in vitro (Patton et al., 1997). Both laminin-1 recently Colognato and Yurchenco (1999) reported that and laminin-2 are present prior to development of purified laminin-2/4 does induce clustering. This result neuromuscular junctions in vivo (Patton et al., 1997). Laminin- prompted us to revisit this question. Using the same 1 appears localized at myotendinous junctions, but it is rarely commercial source of laminin-2/4 as Sugiyama et al. (1997) detected at neuromuscular junctions. In contrast, laminin-2, we also failed to induce AChR clusters and noted that the cells Laminin and α7β1 integrin mediated AChR clustering 2883

A Laminin preincubation 40 Laminin preincubation A 30 Laminin+Agrin No preincubation

25 30 20

20 15

10 AChR clusters/field 10 AChR clusters/field 5

0 0 0 100 200 300 400 0.0 0.1 0.2 0.3 0.4 0.5 Time (mins) Agrin Concentration (nM)

B Laminin preincubation B 30 Laminin+Agrin 12 Laminin preincubation No Preincubation

10 y = -1.9500 + 0.22678x R1 = 0.912 20 8

6 y = -8.1083 + 0.21894x 10 R2 = 0.876 4 AChR clusters/field AChR clusters/field

2

0 0 20 40 60 80 100 120 140 0 2 4 6 8 10 Time (mins) Agrin Concentration (pM) Fig. 6. Preincubation with laminin accelerates AChR cluster Fig. 5. Preincubation with laminin lowers the concentration of agrin formation. C2C12 myotubes were incubated with laminin for 2 required to initiate AChR clustering. C2C12 myotubes were hours. Unbound laminin was then removed and 0.2 nM agrin was preincubated with 60 nM laminin-1 for 2 hours and unbound laminin added. AChR cluster formation was determined over a period of 6 was then removed. 0 to 0.5 nM was then added for 2 hours and hours. Alternatively, both 0.2 nM agrin and 60 nM laminin were number of AChR clusters determined. (A) Preincubation with added together and AChR clusters were determined over the same laminin-1 increased the number of AChR clusters that form even at time course. (A) Preincubation with laminin reduced the lag phase low concentrations of agrin compared to induction with agrin alone. for AChR clustering. The mean values scored in triplicate samples ± (B) Preincubation with laminin lowers by 20-fold the concentration s.e., are given. (B) The rates of AChR cluster formation, determined of agrin needed to induce comparable numbers of AChR clusters. by linear regression analysis, over the period of linear increase were The mean values scored in triplicate samples ± s.e., are given. identical. Thus, preincubation with laminin lowers the threshold concentration of agrin needed to promote clustering and minimizes the lag period, but does not influence the rate of cluster formation. appeared ‘unhealthy’ and loosely attached. We therefore dialyzed the laminin to remove potential toxic components. The dialyzed laminin-2/4 did promote AChR clustering and co- localization with the α7 integrin (Fig. 8). An independent DISCUSSION preparation of laminin purified from human placenta also induced clustering. Laminin-2/4 also enhanced agrin-induce The neuromuscular junction develops through an orchestrated AChR aggregation. Purified laminin-9 and -11 were not recruitment of proteins to the site of motor neuron innervation available to test. Thus in addition to laminin-1, isoforms of (Lichtman and Sanes, 1999). Central to the formation of this laminin that are localized at the neuromuscular junction, will structure is the clustering of acetylcholine receptors to the post- promote AChR clustering. synaptic membrane. One developmental trigger for this event 2884 D. J. Burkin and others

B 100

80

60

40

Fig. 7. The cytoplasmic domain of α7 integrin chain regulates Percent Colocalization 20 colocalization of the integrin with AChR clusters. A common tyrosine residue in the α7A and α7B cytoplasmic domains were 0 mutated to phenylalanine (YTF) and a threefold DXHP repeat in the Agrin Laminin-1 Merosin α7B cyoplasmic domain was deleted. C2C12 myotubes transfected to express the wild-type rat α7AX2, α7BX2 and mutant α7AX2- C YTF, α7BX2-YTF and α7BX2-DXHP integrins were induced with 40 60 nM laminin, 0.5 nM agrin, or both 60 nM laminin and 0.5 nM agrin for 16 hours. AChR clusters were visualized with rhodamine 30 labeled bungarotoxin and colocalization of the integrin with AChR clusters was determined with O26 monoclonal antibody and FITC- labeled anti-mouse antibody. Induction with laminin or agrin and 20 laminin promotes a high level of colocalization of the integrin with

AChR clusters. Induction with agrin fails to promote colocalization 10 α of wild-type 7 chain isoforms with AChR clusters but does promote AChR clusters/field colocalization with both the α7AX2-YTF and α7BX2-YTF mutants. Thus the α7 chain cytoplasmic domain negatively regulates the 0 localization of the integrin with agrin-induced AChR clusters. The Agrin

mean values scored in triplicate samples ± s.e., are given. Merosin Laminin-1 Merosin+Agrin Laminin+Agrin is the proteoglycan neural agrin that is synthesized and secreted by the innervating motor neuron. Several lines of in vitro evidence now suggest that laminin and agrin play an Merosin+Laminin-1+Agrin integral role in the development of this junctional site. (1) Fig. 8. Merosin (laminin-2/4) induces AChR clustering and Laminin can independently induce AChR clusters (Sugiyama colocalization with the α7β1 integrin. C2C12-α7BX2 myofibers et al., 1997; Montanaro et al., 1998; Burkin et al., 1998). were induced with 0.5 nM agrin and/or 60 nM laminin-1 or 90 nM However, together agrin and laminin promote a more vigorous merosin for 16 hours. (A) Immunofluorescence localization of the rat α7 integrin (green) with laminin-2/4-induced AChR clusters (red). clustering response than either inducer alone (Sugiyama et al., (B) The α7β1 integrin is colocalized with merosin and laminin-1 1997, Burkin et al., 1998). (2) Induction with laminin or with induced AChR clusters but not with agrin-induced clusters. agrin and laminin, promotes the physical association and (C) Enhanced cluster formation upon induction with agrin and either colocalization of the α7β1 integrin and acetylcholine receptor. laminin-1 or merosin. (3) Crosslinking the muscle α7β1 laminin receptor with anti- α7 antibody promotes AChR aggregation, whereas blocking this integrin inhibits laminin-induced clustering (Burkin et al., chain cytoplasmic domain alter agrin-induced colocalization of 1998). Concentrations of anti-α7 that crosslink the integrin the AChR and integrin. Whereas both mutant and wild-type promote AChR clustering whereas excess antibody and Fab integrin engage in laminin-induced coclustering, only the fragments do not. (4) Laminin is required for the localization tyrosine mutants engage in agrin-induced coclustering. The of specific isoforms of the α7β1 integrin with AChR clusters. molecular basis underlying this change is yet to be determined. Only those isoforms of the α7 integrin chain (α7X2A and CHO cells expressing both mutant and wildtype integrin α7X2B) that are present at neuromuscular junctions in vivo isoforms bind to and migrate on laminin (W. Wang and S. J. (Martin et al., 1996) function in laminin-induced aggregation Kaufman, unpublished results), thus the mutants can interact of AChRs (Burkin et al., 1998). (5) Mutations in the α7 integrin with and respond to laminin. (6) Clusters of AChRs do form Laminin and α7β1 integrin mediated AChR clustering 2885 in agrin deficient mice, albeit they are reduced in size, number Although laminin-1 and laminin-2/4 are present prior to and density (Gautam et al., 1996). The formation of clusters in development of neuromuscular junctions (Patton et al., 1997), the absence of agrin suggests that a co-inducer participates in it is not known which isoform functions in vivo at this stage receptor aggregation: laminin appears to be this co-inducer. of synapse formation. The absence of α1 laminin chain at To further understand the role of laminin and the α7β1 NMJs (Martin et al., 1996) suggests that laminin-2/4 either integrin in the development of the neuromuscular junction, we induces clustering in vivo or rapidly replaces laminin-1. It have examined the dynamics of colocalization of the integrin should be noted that there is not complete agreement on and AChRs during cluster formation. Within 2 hours of whether α1 laminin chain is expressed by skeletal muscle induction with either laminin, or agrin and laminin, both the (Schuler and Sorokin, 1995). Thus laminin-2/4 appears to be α7AX2 and α7BX2 isoforms localized with AChR into the stronger candidate to participate in AChR clustering in clusters. High levels of colocalization of the integrin and AChR vivo. Since laminin-8 (α4β1γ1) may compensate for the clusters were maintained in the presence of laminin. These absence of α2 laminin chains in dy/dy mice (Patton et al., results suggest the α7β1 integrin has an early role in the 1997), it too may serve to induce AChR clustering. development of AChR clusters and this role is dependent on Muscle fibers are surrounded by an extracellular matrix that laminin. Whereas laminin is sufficient to promote is essential to muscle function and integrity. This matrix is colocalization, both laminin and agrin are essential to vigorous present before innervation of the fibers. Laminin, a component cluster formation. In the presence of agrin alone, little of this matrix is therefore present before neural agrin (Patton colocalization of the integrin with AChR clusters was observed et al., 1997). This suggested that the early association of over 16 hours and the number of clusters was comparatively myofibers with laminin might prime these cells and thereby low. In the presence of laminin and absence of agrin the integrin underlie the enhanced response to agrin seen in vitro. To was localized to all clusters, although the numbers and rate of determine if laminin can prime AChR clustering, C2C12 cluster formation was reduced. Thus laminin is necessary for myotubes were preincubated with laminin prior to the addition localization of the integrin to post-synaptic sites and agrin is of agrin. This preincubation enhanced AChR cluster formation essential to rapid, vigorous clustering. ‘Spontaneous’ clustering 2.4 fold, lowered the concentration of agrin required to initiate of AChRs, in the absence of exogenous inducer, may result from AChR clustering 20-fold, and minimized the lag period the synthesis of laminin by myotubes in culture. between cluster formation subsequently induced by agrin. Immunoprecipitation of AChR clusters shows that Laminin can therefore act to prime fibers, enabling them to colocalization of the integrin and AChR is paralleled by a rapidly respond to low concentrations of agrin. laminin-dependent physical interaction between the integrin Although the mechanism of such priming is not known it and AChR. This physical association occurs 2-4 hours after may very well involve integrin-mediated organization of the induction with laminin or laminin and agrin. However, this extracellular matrix on myofibers (Colognato et al., 1999) association is not dependent on cluster formation as it is and/or the physiologic activation of these cells. Physiologic equivalent upon vigorous clustering promoted by laminin and activation may reflect recruitment of additional proteins to agrin and minimal cluster formation induced by laminin alone. form presynaptic complexes that will subsequently engage in In the presence of both laminin and agrin these receptors form AChR clustering (Furher et al., 1999), consolidation of physically stable complexes with each other and aggregate complexes with the cell cytoskeleton, and activation of the within 2 hours. In the absence of agrin, cluster formation is intracellular release of Ca+2 that is essential to clustering minimal but stable physical interactions between the integrin (Megeath and Fallon, 1998; Wallace, 1988). Notably, laminin and AChR form within the same time frame as with both and crosslinking of the α7β1 integrin also promote an increase inducers. Thus, the physical interaction of the integrin with in intracellular calcium (Kwon et al., 2000). α-Dystroglycan AChRs and their aggregation into clusters appear to be separate can bind laminin and agrin and thus may also participate in sequential events in the same pathway. laminin-induced activation of clustering (Bowe and Fallon, Agrin alone does not promote coclustering or a physical 1995; Montanaro et al., 1998; Ruegg and Bixby, 1998). association between the α7β1 integrin and AChR. Interestingly, Although a detailed analysis of the neuromuscular junction this appears to be dependent on the single tyrosine common to in α7 deficient humans and mice (Hayashi et al., 1998; Mayer all α7 cytoplasmic domains (Song et al., 1993). Whereas wild- et al., 1997) has not been reported, the myopathy and delayed type α7A and α7B integrin do not colocalize with AChR motor milestones seen in patients with mutations in their α7 clusters induced by agrin, mutant α7 chains, where the genes may reflect the role of the α7β1 integrin in the formation cytoplasmic tyrosine was mutated to phenylalanine, do and integrity of neuromuscular junctions. colocalize with AChRs. Thus the α7 cytoplasmic domain appears to regulate the integrin’s association with AChRs. We thank Dr Stephen Hauschka for providing the MCK promoter Only specific isoforms of laminin promote AChR clustering. used in these experiments and Dr James Campanelli for the CHO cells Laminin-1 and laminin-2/4 (this paper), but not laminin-11 transfected to express neural agrin. This work was supported by grants (Sugiyama et al., 1997), induce clustering in vitro. Whereas from the Association of America and the National Institutes of Health. Sugiyama et al. 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