Rapsyn Is Required for Musk Signaling and Recruits Synaptic Components to a Musk-Containing Scaffold

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Neuron, Vol. 18, 623±635, April, 1997, Copyright 1997 by Cell Press Rapsyn Is Required for MuSK Signaling and Recruits Synaptic Components to a MuSK-Containing Scaffold Elizabeth D. Apel,* David J. Glass,² Lisa M. Moscoso,* extrasynaptic areas. Several molecules have been impli- George D. Yancopoulos,² and Joshua R. Sanes* cated in this process, based on their distributions in vivo *Departments of Molecular Biology and Pharmacology and their activities in cultured cells. Recently, studies of and of Anatomy and Neurobiology knockout mice have demonstrated crucial roles for three Washington University School of Medicine of them: AChR clustering, as well as numerous other St. Louis, Missouri 63110 aspects of postsynaptic differentiation, are grossly defi- ² Regeneron Pharmaceuticals, Inc. cient in the muscles of mice lacking agrin (Gautam et 777 Old Saw Mill River Road Tarrytown, New York 10591 al., 1996), MuSK (DeChiara et al., 1996), or rapsyn (Gau- tam et al., 1995). Agrin was identified as a factor capable of inducing Summary aggregation of AChRs already present in the plasma membrane of cultured muscle cells. In vivo, agrin is Agrin-induced clustering of acetylcholine receptors synthesized by motoneurons, transported down motor (AChRs) in the postsynaptic membrane is a key step axons, released from nerve terminals, and incorporated in synaptogenesis at the neuromuscular junction. The into the basal lamina of the synaptic cleft. Muscles also receptor tyrosine kinase MuSK is a component of the produce agrin, but alternatively spliced isoforms ex- agrin receptor, while the cytoplasmic protein rapsyn pressed by motoneurons are .1000-fold more active is necessary for the clustering of AChRs and all other than those expressed by muscle (reviewed by McMa- postsynaptic membrane components studied to date. han, 1990; Bowe and Fallon, 1995). Thus, agrin appears We show here that MuSK remains concentrated at to be a nerve-derived organizer of AChR clustering. synaptic sites in rapsyn-deficient mutant mice, sug- MuSK is a transmembrane tyrosine kinase that is se- gesting that MuSK forms a primary structural scaffold lectively expressed by skeletal muscle and is concentrated with AChRs in the postsynaptic membrane of to which rapsyn attaches other synaptic components. adult neuromuscular junctions (Valenzuela et al., 1995). Using nonmuscle cells, we show that rapsyn±MuSK Myotubes cultured from MuSK mutant mice express interactions are mediated by the ectodomain of MuSK, normal levels of AChRs but are unresponsive to agrin suggesting the existence of a transmembrane inter- (Glass et al.,1996). Moreover, MuSK is rapidly phosphor- mediate. In addition to rapsyn's structural role, we ylated in response to agrin, and introduction of a domi- demonstrate that it is required for an early step in nant negative mutant MuSK into muscle cells inhibits MuSK signaling, AChR phosphorylation. This signaling agrin's ability to induce AChR clustering (Glass et al., requires the kinase domain of MuSK, but not its ecto- 1996; D. J. G., E. D. A., J. R. S., and G. D. Y., unpublished domain. Thus, MuSK may interact with rapsyn in multi- data). Agrin does not bind detectably to purified MuSK ple ways to play both structural and signaling roles in but can be chemically cross-linked to MuSK on the myo- agrin-induced differentiation. tube surface, suggesting that MuSK interacts with additional components, termed MASC (Glass et al., 1996). Thus, MuSK appears to be a critical part of a multicom- Introduction ponent signal transducing receptor complex for agrin. Rapsyn (previously called 43K protein) is a cyto- One of the most intensively studied steps in the forma- plasmic peripheral membrane protein that is precisely tion of a synapse is the accumulation of nicotinic acetyl- colocalized with AChRs in vivo (Froehner et al., 1981; choline receptors (AChRs) in the postsynaptic mem- Sealock et al., 1984), can be cross-linked to AChRs brane of the skeletal neuromuscular junction (reviewed (Burden et al., 1983), and is present at embryonic AChR by Hall and Sanes, 1993). In uninnervated myotubes, clusters as soon as they form (Noakes et al., 1993). AChRs are evenly distributed in the plasma membrane Removal of rapsyn from AChR-rich membranes in- at a density of z1000/mm2. As synapse formation pro- creases the rotational mobility of the AChRs (Barrantes ceeds, the concentration of AChRs reaches 10,000/mm2 et al., 1980; Rousselet et al., 1982), and coexpression in the postsynaptic membrane and falls to z10/mm2 in of rapsyn with AChRs in nonmuscle cells induces clus- extrasynaptic areas (Fertuck and Salpeter, 1974; Bevan tering of the latter (Froehner et al., 1990; Phillips et al., and Steinbach, 1977). The nerve induces this redistribu- 1991a). Conversely, no AChR clusters form in muscles tion by providing several signals that trigger a combina- cultured from rapsyn mutant mice, even in the presence tion of transcriptional and posttranslational regulatory of saturating amounts of agrin (Gautam et al., 1995). mechanisms within the muscle (reviewed by Duclert and Thus, rapsyn appears to be an essential component of Changeux, 1995). During the past few years, there has the cytoskeletal apparatus responsible for agrin-induced been considerable progress in identifying the nerve- AChR clustering. Interestingly, however, neuromuscular derived signals and the intramuscular mechanisms re- junction formation is not as drastically perturbed in mice sponsible for this aspect of synaptogenesis (reviewed lacking rapsyn as in mice lacking agrin or MuSK, sug- by Apel and Merlie, 1995a; Sanes, 1997). gesting that rapsyn may be required for only a subset Here, we focus on the nerve-induced aggregation of of agrin/MuSK-activated pathways. For example, sub- AChR in the muscle membrane, which leads to their synaptic myonuclei selectively transcribe AChR genes concentration at synaptic sites and their depletion from in rapsyn-deficient mice, but not in mice lacking agrin Neuron 624 or MuSK (Gautam et al., 1995; DeChiara et al., 1996; as previously reported, MuSK remained localized at syn- Gautam et al., 1996). These studies have defined a path- aptic sites in adults (not shown). Thus, the synaptic way for clustering in which agrin is the signal, MuSK is localization of MuSK protein observed in adults is part of the signaling receptor, and rapsyn is part of the achieved well before birth, and MuSK is present at the effector mechanism. It now becomes important to define earliest detectable embryonic AChR clusters. the interactions of agrin with MuSK and of MuSK with Next, we assessed the distribution of MuSK in mice rapsyn. In this paper, we analyze the structural and func- lacking rapsyn. Because AChRs and other postsynaptic tional relationship between MuSK and rapsyn. We show membrane proteins do not cluster at synapses in this mu- that MuSK is concentrated at neuromuscular junctions tant, we marked synaptic sites with antibodies to SV-2, in rapsyn-deficient mice, indicating that neither rapsyn a component of the synaptic vesicles that accumulate nor AChRs regulate the distribution of MuSK. In this in nerve terminals (Gautam et al., 1995). Surprisingly, respect, MuSK differs from all other membrane and cy- MuSK was highly concentrated at synapses in mutant toskeletal components of the postsynaptic apparatus mice (Figure 1B). This finding demonstrates that MuSK studied to date, which fail to accumulate at synaptic can aggregate by a rapsyn-independent mechanismand sites in the absence of rapsyn (Gautam et al., 1995; raises the possibility that MuSK nucleates assembly of Moscoso et al., 1995b). Thus, MuSK may be part of a the postsynaptic apparatus, perhaps serving as a pri- primary scaffold, which then requires rapsyn to recruit mary scaffold to which additional components are sub- AChRs and other proteins to the synapse. We use a sequently recruited. nonmuscle cell system to show that rapsyn can indeed In adult muscle, mRNAs encoding several synaptic link AChRs and other synaptic components to MuSK. proteins, including AChRs (Merlie and Sanes, 1985; Surprisingly, however, this association is mediated by Moscoso et al., 1995a) and MuSK (Valenzuela et al., the extracellular domain of MuSK, suggesting the exis- 1995), are themselves concentrated at synapses. For tence of an intermediary transmembrane component AChRs, synaptic localization of the mRNA reflects pref- that links MuSK to rapsyn. Finally, we examine agrin- erential transcription of AChR subunit genes by syn- induced signaling events in rapsyn-deficient myotubes apse-associated myonuclei (Sanes et al., 1991; Duclert and find that agrin-induced MuSK activation occurs nor- and Changeux, 1995). The preferential transcription of mally, whereas MuSK-dependent phosphorylation of AChR subunit genes by synaptic nuclei persists in the AChRs does not occur and is thus rapsyn-dependent. absence of rapsyn (Gautam et al., 1995) but is abolished Together, these results suggest that the intra- and extra- in the absence of MuSK (DeChiara et al., 1996). Since cellular domains of MuSK interact with rapsyn in distinct synaptic localization of the MuSK and AChR proteins ways. We propose that both interactions are required are regulated differentlyÐthe former being rapsyn inde- for agrin-dependent postsynaptic differentiation. pendent and the latter rapsyn dependentÐwe used in situ hybridization to assess the distribution of

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