Signals mediating ion channel clustering at the neuromuscular junction

357

Signals mediating junction Marcie Colledge High densities channels folds,

respectively,

Clustering

and Stanley C Froehner*

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in the crests

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Addresses Department of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7545, USA *e-mail: [email protected] Current Opinion

in Neurobiology

ensure rapid, efficient synaptic transmission and initiation of action potentials in the postsynaptic cell. Understanding the mechanisms involved in forming synaptic specializations remains a key question in neurobiology. This review will consider the signals involved in AChR and NaCh clustering during synaptogenesis, focusing on agrin, the receptor complex that it activates and the role of tyrosine phosphorylation in this process. The involvement of rapsyn and syntrophin, cytoplasmic proteins associated with AChRs and NaChs, respectively, will be evaluated.

of the structural clustering,

the most surprising and utrophin

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in receptor

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1998, 8:357-363

http://biomednet.com/elecref/0959438800800357 0 Current Biology Ltd ISSN 0959-4388 Abbreviations AChR acetylcholine receptor AChR-inducing activity ARIA green fluorescent protein GFP mitogen-activated protein MAP MASC MUSK accessory specificity component muscle-specific kinase MUSK sodium channel NaCh domain in PSD-95, discs large and zona occludens 1 (ZO-1) PDZ receptor-associated protein at synapses rapsyn rapsyn-associated transmembrane link RATL receptor tyrosine kinase RTK Src homology SH tetratricopeptide re’peat TPR

Introduction Signalling in the nervous system depends on a highly ordered and regulated arrangement of proteins, particularly ion channels, at the synapse. At the vertebrate neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are clustered at high density on the crests of the postsynaptic folds in precise register with presynaptic sites of acetylcholine release [l]. In the depths of the postjunctional folds, immediately adjacent to these receptor clusters, voltage-activated sodium channels (NaChs) are present at a density ten times that on the nonsynaptic membrane [2,3]. Together, these remarkable distributions

Clustering of acetylcholine

receptors

Current evidence indicates that the high concentration of AChRs at the synapse arises, in part, from the selective transcription of AChR subunit genes in specialized synaptic nuclei. The nerve-derived molecule ARIA (AChR-inducing activity) [4], a member of the neuregulin family of ligands, is thought to induce AChR gene expression via activation of the ErbB receptor tyrosine kinases (RTKs; recently reviewed in [S]). There is then the problem of accumulating and anchoring these receptors in the postsynaptic membrane. Agrin is a signalling molecule essential for this process. Originally identified for its striking ability to cause the aggregation of AChR when applied to cultured muscle cells, agrin is synthesized and secreted by motor neurons and becomes incorporated into the synaptic basal lamina [6,7]. Ectopic expression of recombinant agrin in adult muscle induces the formation of postsynaptic-like structures that closely resemble the muscle endplate [8*-lo’]. The muscle-specific RTK MUSK is a critical component of agrin’s signalling receptor complex ([ll]; for recent review see [12]). Mice deficient in either agrin or MUSK fail to form neuromuscular synapses and do not survive past birth [13,14].

Agrin and MUSK On many levels, agrin and MUSK are atypical of conventional growth factor-RTK systems. Whereas ligands for RTKs are characteristically small peptide growth factors, agrin is a large, multidomain heparan sulfate proteoglycan [15,16], which itself may be capable of binding growth factors [17]. Agrin does not bind directly to MUSK; binding and subsequent activation appear to require an unidentified muscle-specific co-receptor, MASC (MUSK accessory specificity component) [ll]. It is now apparent that there are a multitude of binding sites for agrin on the muscle cell, including a-dystroglycan, a dystrophin-associated protein [18-211, neural cell adhesion molecule [16], heparin-binding growth associated molecule [17], laminin [ZZ] and certain integrins [23]. It remains to be tested whether any of these correspond to MASC. In contrast to all other RTK signalling systems, in which the biological

358

Signalling mechanisms

response can be wholly accounted for by activation of the catalytic domain of the receptor, activation of MUSK’S kinase domain is insufficient to mediate AChR clustering [24**]. In fact, the extracellular domain of MUSK appears to be required to couple MUSK to its downstream effector rapsyn, an intracellular protein closely associated with the AChR. This surprising observation implicates the existence of a transmembrane protein (RATL, rapsyn-associated transmembrane link) that serves to link MUSK to rapsyn [24**,25**]. The identification of MASC and RATL may further differentiate this system from other characterized growth factor signalling systems.

Tyrosine phosphorylation of the AChR Agrin stimulation of cultured myotubes induces a rapid but transient increase in p-subunit tyrosine phosphorylation that precedes AChR clustering, suggesting that this may be a critical, and perhaps a prerequisite, signal in the clustering pathway [26,27]. The stimulation of myotubes expressing chimeric MUSK-TrkC receptors (containing the ectodomain of TrkC fused to the catalytic domain of MUSK) with the surrogate ligand neurotrophin 3 (NT3), causes p-subunit phosphorylation [24”], indicating that MUSK kinase activation is sufficient to mediate this signalling event. Notably, MUSK-induced P-subunit phosphorylation fails to occur in rapsyn-deficient myotubes. One possibility is that agrin-activated MUSK directly phosphorylates the p subunit. In fact, complexes containing MUSK and the AChR can be purified from cultured muscle cells and agrin-induced phosphorylation of MUSK is correlated with B-subunit phosphorylation [28’]; however, the treatment of myotubes with the tyrosine kinase inhibitor staurosporine prevents agrin-induced phosphorylation of the p subunit without blocking MUSK activation, indicating that MUSK activation can be uncoupled from AChR phosphorylation. Thus, the B subunit does not appear to be a direct substrate for MUSK [28*]. An alternative model is that agrin-stimulated MUSK activates a second kinase, which in turn acts directly on the p subunit. One potential candidate is the nonreceptor tyrosine kinase Src. Fuhrer and Hall [29’] recently reported that Src binds to and phosphorylates a fusion protein corresponding to the intracellular loop of the p subunit containing the conserved tyrosine phosphorylation site. AChR complexes isolated from cultured muscle cells contain both Src and the related kinase Fyn [29*]. Fyn association with the AChR appears to be phosphorylation dependent [30,31], leading to a model in which Src first binds to and phosphorylates the p subunit and in doing so creates a binding site for Fyn [29*]. The functional consequence, if any, of S-subunit phosphorylation is not clear at present. The fast calcium chelator BAPTA prevents agrin-induced AChR clustering in cultured myotubes, but does not inhibit P-subunit phosphorylation [32-l. In addition, although MUSK catalytic

activity is sufficient to induce o-subunit phosphorylation, it is not sufficient to induce AChR clustering [24”]. Clearly then, P-subunit phosphorylation alone does not trigger AChR clustering. But is it necessary? Two lines of evidence imply that it may not be. First, two recent reports demonstrate that laminin can induce AChR clustering via a signalling pathway that is independent of agrin and MUSK [33*,34*]. Treatment of myotubes with laminin does not cause phosphorylation of either MUSK or the AChR 0 subunit, indicating that, at least in this signalling pathway, AChR phosphorylation is not a prerequisite for clustering. Second, in heterologous cells, mutation of the conserved tyrosine phosphorylation site in the p subunit does not prevent rapsyn-induced clustering [35,36]. Whether this site is critical for agrin-mediated AChR clustering in ojwo, however, remains to be tested. In addition to the p subunit, the y and 6 subunits of the AChR are also targets of tyrosine kinases (reviewed in [37]). Recent evidence suggests that tyrosine phosphorylation of the 6 subunit also may be an important mediator of signalling at the postsynaptic membrane. The Ras effector Grb2 -a modular adaptor protein consisting of one Src homology 2 (SH2) and two SH3 domains-forms a complex with the Torpedo AChR [38*]. Direct binding is mediated by a high-affinity interaction between the tyrosine phosphorylated 6 subunit and the SH2 domain of Grb2. Dephosphorylation of the AChR prevents Grb2 binding, suggesting that phosphorylation of the 6 subunit regulates the interaction, and may potentially trigger the activation of the Raslmitogen-activated protein (MAP) kinase signal transduction pathway. This is especially intriguing given recent reports that ARIA induces the synapse-specific transcription of AChR genes via activation of the Ras/MAP kinase and phosphatidylinositol 3-kinase pathways [39-41].

Ra psyn The intracellular peripheral membrane protein rapsyn (43 kDa protein), plays a crucial role in clustering AChR. In W&JO,rapsyn and AChR are distributed co-extensively in the postsynaptic membrane 1421. When expressed in heterologous cells, rapsyn forms membrane clusters and is capable of recruiting AChR to these clusters in an agrin-independent manner [43,44]. Like agrin and MUSK knockout mice, rapsyn-deficient mice fail to form neuromuscular synapses [45]. Although agrin stimulation of rapsyn-deficient myotubes leads to MUSK activation, the AChR /3 subunit does not become phosphorylated and AChR clustering fails to occur [25”,45]. Thus, rapsyn is required to couple agrin-mediated MUSK activation to AChR phosphorylation and, more importantly, is an essential downstream component in agrin’s clustering pathway. In heterologous expression systems, rapsyn is capable of inducing MUSK clustering (a process that, surprisingly, requires the ectodomain of MUSK) [25’*,46]. This observation led Gillespie et al. [46] to propose that rapsyn may be required for clustering MUSK at

Signals mediating ion channel clustering at the neuromuscular junction Colledge and Froehner

the developing postsynaptic membrane in viva; however, MUSK is the only postsynaptic protein so far examined that remains clustered at the synapse in rapsyn-deficient mice [ZS”]. Thus, in vtio, rapsyn is likely to function in the recruitment of other postsynaptic proteins to a pre-existing, primitive MUSK-based scaffold [25**]. In agreement with this conclusion, some aspects of postsynaptic differentiation, including synapse-specific transcription, which are lost in MUSK-deficient mice, are preserved in the rapsyn knockouts [14,45]. This supports the notion that agrin stimulation of MUSK initiates several signalling cascades that lead to the formation of the postsynaptic apparatus and that rapsyn is required for only a subset of these (for a recent review, see [12]). Rapsyn is composed of several conserved protein domains: an amino-terminal myristoylation site [47], eight tandem tetratricopeptide repeats (TPRs) [48] and a carboxy-terminal zinc ring finger domain [49,50], which is immediately followed by a consensus site for serine phosphorylation. TPRs are 34 amino acid repeats believed to form amphipathic cx helices that mediate protein interactions. Recently, a major step forward in our understanding of the function of rapsyn has come from the expression of AChRs with chimeric proteins containing various domains of rapsyn fused to green fluorescent protein (GFP) [Sl”]. The myristoylated amino-terminal 15 amino acids of rapsyn are sufficient to mediate membrane targeting. A rapsyn-GFP chimera containing the first two TPRs forms membrane clusters, suggesting that TPR domains function to mediate rapsyn self-association; however, this chimera, as well as one containing TPRs 1-7, is unable to cluster AChR. Interaction with the AChR appears instead to depend on a sequence within TPR 8 that has a high probability of forming a coiled-coil structure. The insertion of mutations in rapsyn that disrupt coiled-coil propensity prevent AChR clustering [Sl’*]. The emerging picture of this essential protein is one in which certain TPRs mediate self-association of rapsyn, whereas a coiled-coil domain mediates interaction with AChRs. The targeting of other proteins to the postsynaptic membrane could occur by interaction with other TPRs and the ring finger.

Clustering of sodium channels NaChs are found throughout the sarcolemma but are concentrated at least tenfold at the synapse where they are restricted to the depths of the postjunctional folds and may be associated with ankyrin [2,3]. Synaptic clusters of NaChs form after birth in the rat, or approximately two weeks later than AChR clusters are first detected [Xl. Despite this difference, current evidence indicates that agrin is also the stimulus that causes NaCh clustering. Cultured adult muscle fibers, when stimulated locally with agrin secreted from COS cells, develop NaCh clusters at the site of contact between the two cells [531. Ectopic synaptic specializations formed by local expression of recombinant agrin in muscle in vtio also contain high densities of NaChs (9.1. Whether MUSK is involved in this

359

process is not yet known. It is interesting to note that one highly active isoform of agrin is expressed at its highest levels after birth [54] and thus may be especially relevant to NaCh clustering [53]. Figure 1

(a) AChR clustering

Fyn Grb2

MASC Szc\#? RATL Agrin I, MUSK +Rapsyn+AChR-pY

LamininI,

+

ca*+ +

a-dystroglycan

AChR clustering

&’

(b) NaCh clustering 7 Agrin *Agrin

receptor’l,

7 SyntrophinlankyrinI,

NaCh clustering

CurrentO!ainion in Neurobiolwv

Signalling events leading to AChR and NaCh clustering. (a) Agrin initiates a signalling cascade by activating MUSK, an event that requires the co-receptor MASC. MUSK is connected to rapsyn by the so-called transmembrane linker RATL Tyrosine phosphorylation (pv) of the AChR requires both MUSK catalytic activity and rapsyn. The kinase responsible for AChR phosphorylation has not been identified, but Src is a potential candidate. Fyn and GrbP may then bind the tyrosine-phosphorylated receptor, possibly initiating other signalling events. Whether AChR phosphorylation is critical for clustering is not yet clear. A calcium-regulated step, which occurs either downstream or in parallel to AChR phosphorylation, appears to be important for clustering. Laminin can also cause AChR clustering in a manner that is independent of agrin and MUSK, but appears to require a-dystroglycan (see text for details). (b) Fewer details of the NaCh clustering pathway are known. Like AChR clustering, agrin is the stimulus that initiates NaCh clustering, presumably through interaction with a specific receptor. Syntrophin and ankyrin are involved in targeting NaChs to the dystrophin complex and the actin cytoskeleton, potentially clustering them in the troughs of the folds (see text for details).

Syntrophins Compared to that of AChR clustering, our knowledge of the signalling processes underlying NaCh clustering is sketchy, Recently, however, a link between skeletal (and cardiac) muscle NaChs and the dystrophin complex was established [%*I. Syntrophins are dystrophin-associated modular proteins comprising two pleckstrin homology domains, a PDZ domain and a unique sequence [56,57]. Dystrophin is directly linked, via its amino-terminal region, to cortical actin and, via a carboxy-terminal site, to the transmembrane dystroglycan complex, which interacts with extracellular matrix through agrin/laminin (see [58] for a recent review). The two forms of NaChs expressed in skeletal muscle have carboxy-terminal amino acid sequences (S/TXV) that match the consensus for binding to PDZ domains [59-61]. Indeed, both bind to syntrophin PDZ domains [55*,62*] and, more importantly, NaChs can be specifically immunopurified with the dystrophin complex [SS’]. Thus, syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via association

360

Signalling mechanisms

with the dystrophin complex and may play a role similar to that of rapsyn in AChR clustering.

Dystrophinbtrophin Complexes of proteins associated with dystrophin are highly concentrated at the neuromuscular junction. Utrophin, a close relative of dystrophin, is highly restricted at the synapse and is distributed identically to AChR and rapsyn at the crests of the postjunctional folds. This distribution implied that utrophin might be critical to AChR clustering. Surprisingly, utrophin-deficient mice exhibit subtle defects in their neuromuscular synapses, with only a small reduction in the density of AChRs (63,641. Double mutants lacking both utrophin and dystrophin show enhanced dystrophic symptoms compared to either mutant alone, but the synapses are nearly normal, at least in terms of clustered AChRs and gross morphology [65”,66**]. To date, the clustering of NaChs has not been examined in these mice, but studies of these and other mutants lacking components of the dystrophin complex may prove useful in understanding the regulation of NaCh clustering at the neuromuscular synapse.

Conclusions Despite significant progress in our understanding of the signals that lead to the clustering of ion channels at the neuromuscular junction (see Figure l), many aspects of these events remain obscure. The specific interactions that connect agrin and MUSK need further characterization. This will require the identification of MASC, as well as other potential ligands of MUSK, which may include a more conventional growth factor-like protein. Similarly, the identification of RATL should provide insight into the mechanism by which MUSK activation is linked to rapsyn-dependent AChR clustering. Aside from AChR phosphorylation, the functional consequence of which is unclear, we know essentially nothing of the downstream intracellular signals that culminate in receptor clustering. Studies focused on identifying other postsynaptic proteins that associate with rapsyn may prove particularly enlightening. Compared to AChR clustering, we understand even fewer of the events that lead to NaCh clustering in response to agrin. It will be interesting to determine whether this pathway also involves MUSK. Given rapsyn’s location at the tops, rather than the bottoms of the postsynaptic folds, it seems unlikely that it plays

Figure 2

Agrin

Lamininlagrin

MASC

Crest

:.

Lamininlagrin

Trough Current Opinion I” Neurobiology

Molecular specializations in the neuromuscular postsynaptic membrane. Distinct molecular complexes are localized on the crests and in the troughs of the postjunctional folds. AChR, rapsyn and utrophin are clearly localized to the crests of the folds. Because MUSK is linked to rapsyn via RATL, we have placed the MUSK-MA% complex in this same region, although definitive evidence for this localization is lacking. NaCh, dystrophin and ankyrin (Ank) have all been shown by ultrastructural studies to be localized to the troughs of the folds. cc-dystroglycan (aDG) and B-dystroglycan (8) are associated with both utrophin and dystrophin and thus are likely to be found throughout the postsynaptic membrane. The isoforms of syntrophin (Syn) on the crests and in the troughs are likely to be different.

Signals mediating ion channel

a direct role in NaCh clustering. Thus, identification of other intracellular targets of agrin will be critical for further characterization of this pathway. A model of the postsynaptic membrane that takes into account the known distributions of signalling and structural proteins is shown in Figure 2.

clustering

at the neuromuscular

junction

Colledge

and Froehner

361

10. .

Cohen I, Rimer M, Lomo T, McMahan UJ: Agrin-induced postsynaptic-like apparatus in skeletal muscle fibers in I&CL MO/ Cell Neurosci 1997, 9:237-253. Together with [8’,9’], this paper demonstrates that when recombinant agrin is ectopically expressed outside of the synapse, it clusters several components of the postsynaptic apparatus, suggesting that agrin is sufficient to cause the induction of synaptic specializations in viva. 11.

Glass DJ, Bowen DC, Stitl TN, Radziejewski C, Bruno J, Ryan TE, Gies DR, Shah S, Mattsson K, Burden SJ et al.: Agrin acts via a MUSK receptor complex. Cell 1996, 85:513-523.

12.

Glass DJ, Yancopoulos GD: Sequential roles of agrin, MUSK and rapsyn during neuromuscular junction formation. Curr Opin Neurobiol 1997, 7~379-384.

13.

Gautam M, Noakes PG, Moscoso L, Rupp F, Scheller RH, Merlie JP, Sanes JR: Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Ce// 1996, 85:525-535.

Understanding the assembly of the neuromuscular junction is certainly important in its own right, but the expectation has been that concepts of neuromuscular synaptogenesis would be applicable to synapse formation between neurons. The finding that neuromuscular proteins such as agrin [l&67], rapsyn [68], and syntrophin [57,69] are expressed in neurons and that a coiled-coil protein, yotiao, is postsynaptically localized at both neuromuscular and central synapses [70] bodes well for this expectation becoming a reality.

14.

DeChiara TM, Bowen DC, Valenzuela DM, Simmons MV, Poueymirou WT, Thomas S, Kinetz E, Compton DL, Rojas E, Park JS et a/.: The receptor tyrosine kinase MUSK is required for neuromuscular junction formation in viva. Cell 1996, 85501-512.

15.

Tsen G, Halfter W, Kroger S, Cole GL: Agrin is a heparan sulfate proteoglycan. J Biol Chem 1995, 270:3392-3399.

Acknowledgements

16.

Burg MA, Halfter W, Cole GJ: Analysis of proteoglycan expression in developing chicken brain: characterization of a heparan sulfate proteoglycan that interacts with the neuronal cell adhesion molecule. J Neurosci Res 1995, 41:49-64.

1 7.

Daggett DF, Cohen MW, Stone D, Nikolics K, Rauvala H, Peng HB: The role of an agrin-growth factor interaction receptor clustering. MO/ Cell Neurosci 1996, 8:272-285.

We thank Jonathan Cohen and Morgan Sheng for sending manuscripts

to publication

and Marvin Adams for helpful comments.

prior

Research

in our laboratory is supported by grants from the National Institutes of Health and the Muscular Dystrophy Association. M Colledge holds a fellowship from

the Royster Society of Fellows.

References

and recommended

. l

.

Fertuck HC, Salpeter MM: Localization of acetylcholine receptor by 12sl-labeled alpha bungarotoxin binding at mouse motor endplates. Proc Nat/ Acad Sci USA 1974, 71 :1376-l 376.

2.

Flucher BE, Daniels MP: Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kDa protein. Neuron 1989, 3:163-l 75. Boudier JL, Le Treut T, Jover E: Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 12sl-a-scorpion toxin. II. Sodium channel distribution on postsynaptic membranes. J Neurosci 1992, 12:454-466.

4.

Jessell TM, Siegel RE, Fischbach GD: Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc Nat/ Acad Sci USA 1979, 76:5397-5401.

5.

Gassmann M, Lemke G: Neuregulins and neuregulin receptors in neural development Curr Opin Neurobiol 1997, 7:87-92.

6.

Godfrey EW, Nitkin RM, Wallace BG, Rubin LL, McMahan UJ: Comoonents of Toroedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J Cell Bioll984, g&615-627.

7.

Nitkin RM, Smith MA, Magill C, Fallon JR, Yung-Mae MY, Wallace BG, McMahan UJ: Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol 1987, 105:2471-2478.

Jones G, Meier T, Lichtsteiner M, Witzemann V, Sakmann B, Brenner HR: Induction by agrin of ectopic and functional postsynaptic-like membrane in innervated muscle. Proc Nat/ Acad Sci USA 1997, 94:2654-2659. See annotation [lo’]. 6. .

Meier T, Hauser DM, Chiquet M, Landmann L, Ruegg MA, Brenner HR: Neural agrin induces ectopic postsynaptic specializations in innervated muscle fibers. J Neurosci 1997, 17:6534-6544. See annotation [lo’].

9. .

Bowe MA, Deyst KA, Leszyk JD, Fallon JR: Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans. Neuron 1994, 12:li 73-1180.

20.

Camoanelli JT. Roberds SL. Camobell KP. Scheller RH: A role for dystrophin-associated’glycdprotein; and utrophin in agrininduced AChR clustering. Cell 1994, 77:663-674.

20.

Gee SH, Montanaro F, Lindenbaum MH, Carbonetto S: Dystroglycan-a. a dystrophin-associated glycoprotein, functional agrin receptor. Cell 1994, 77:675-686.

of special interest of outstanding interest

1.

3.

18.

reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

in ACh

is a

21.

Sugiyama J, Bowen DC, Hall ZW: Dystroglycan muscle agrin. Neuron 1994, 13:103-l 15.

binds nerve and

22.

Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA: Agrin binds to the nerve-muscle basal lamina via laminin. J Cell Biol 1997, 137:671-683.

23.

Martin PT, Sanes JR: lntegrins mediate modulate agrin signaling. Development

adhesion to agrin and 1997, 124:3909-3917.

24. ..

Glass DJ, Apel ED, Shah S, Bowen DC, DeChiara TM, Stitt TN, Sanes JR, Yancopoulos GD: Kinase domain of the musclespecific receptor tyrosine kinase (MUSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: required role for the MUSK ectodomain? Proc Nat/ Acad Sci USA 1997, 9418848-8853. This paper reports that MUSK catalytic activity is required for AChR phosDhorvlatiOnand althouoh necessarvfor AChR clusterina. it is not sufficient. It brovi:desfurther supp& (in additidn to [25**1) for the &portance of MUSK’S extracellular domain in mediating AChR clustering, distinguishing this system from other RTK signalling systems. 25. ..

Apel ED, Glass DJ, Moscoso LM, Yancopoulos GD, Sanes JR: Rapsyn is required for MUSK signaling and recruits synaptic components to a MUSK-containing scaffold. Neuron 1997, 18:623-635. This paper demonstrates that MUSK remains at postsynaptic sites in rapsyndeficient mice and that rapsyn is required to couple agrininduced MUSK activation to AChR phosph&$ation. This paper also implicates the existence of a transmembrane protein, RATL, which links the ectodomain of MUSK to cytoplasmic rapsyn. 26.

Wallace BG, Qu Z, Huganir RL: Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron 1991, 6:869-878.

27.

Ferns M, Deiner M, Hall Z: Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation. J Cell Bid 1996, 132:937-944.

28. .

Fuhrer C, Sugiyama JE, Taylor RG, Hall ZW: Association of muscle-specific kinase MUSK with the acetylcholine receptor in mammalian muscle. EMBO J 1997, 16:4951-4960.

362

Signalling

mechanisms

This paper demonstrates that complexes containing AChRs and MUSK can be isolated from muscle cells and provides evidence that MUSK is not directly responsible for the phosphorylation of the AChR 8 subunit. Fuhrer C, Hall ZW: Functional interaction of Src family kinases with the acetylcholine receptor in C2 myotubes. I Biol Chem 1996.271:32474-32491. ____,_. .._-.. ~_ This paper shows that Src and Fyn kinases co-purify with the AChR and that in vitro, Src can bind and phosphorylate the AChR 6 subunit, supporting a model in which Src directly phosphorylates the receptor, allowing_ Fyn . to bind in a phosphotyrosine-deperrdeni manner. 29. .

30.

31.

Swooe SL. Huaanir RL: Molecular clonina of two abundant pro&in tyrosine kinases in Torpedo electric organ that associate with the acetylcholine receptor. J B/o/ Chem 1993, 26825152-25161. Swope SL, Huganir RL: Binding of the nicotinic acetylcholine receptor to SH2 domains of Fyn and Fyk protein tyrosine kinases. J B/o/ Chem 1994, 269:29817-29824.

Meaeath LJ. Fallon JR: Intracellular calcium reaulates acrin. indiced acetylcholine receptor clustering. J /?eurcsci 1998, 18672-678. The authors show that clamping intracellular calcium levels prevents agrin-induced AChR clustering without blocking AChR phosphorylation, supporting a model in which a calcium-regulated step acts either downstream of, or parallel to, AChR phosphorylation in the clustering pathway.

45.

Gautam M, Noakes PG, Mudd J, Nichol M, Chu GC, Sanes JR, Merlie JP: Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice. Nature 1995, 377~232-236.

46.

Gijlesoie SKH. Balasubramanian S. Funa ET. Huaanir RL: RaosYn clust&s and activates the synapse-sp&iftc receptor tyrosine kinase MUSK Neuron 1998, 16:953-962.

47.

Frail DE, Mudd J, Shah V, Carr C, Cohen JB, Merlie JP: Identification of the mouse muscle 43,000-dalton acetylcholine receptor-associated protein (RAPsyn) by cDNA cloning. J Biol Chem 1988,263:15602-l 5607.

48.

Ponting CCP, Phillips C: Rapsyn’s knobs and holes: eight tetratrico peptide repeats. Biochem J 1996, 314:i 053-l 056.

49.

Freemont PS: The ring finger. A novel protein sequence motif related to the zinc finger. Ann NY Acad Sci 1993, 684:174-l 92.

50.

Scotland PB, Colledge M, Melnikova I, Dai Z, Froehner SC: Clustering of the acetylcholine receptor by the 43-kD protein: involvement of the zinc finger domain. J Cell Biol 1993, 123:71 Q-727

32.

Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW: Laminininduced acetylcholine receptor clustering: an alternative pathway. J Cell Biol 1997, 139:181-l91. See annotation [34*]. 33. .

Montanaro F, Gee SH, Jacobson C, Lindenbaum MH, Froehner SC, Carbonetto S: Laminin and a-dystroglycan mediate acetylcholine receptor aggregation via a MuSKindependent pathway. J Neurosci 1998, 18:i 250-l 260. Together with [33*], this paper provides evidence for a laminin-dependent, agrin-MUSK-independent pathway for AChR clustering. Evidence is provided for a role for a-dystroglycan in this process.

Ramarao MK, Cohen JB: Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn. Proc Nat/ Acad Sci USA 1998, 95:4007-4012. By coexpressing rapsyn-GFP chimeric proteins with the AChR, this study demonstrates that the domains of rapsyn involved in self-clustering and AChR clustering are distinct. 51. ..

52.

Lupa MT, Krzemien DM, Schaller KL, Caldwell JH: Expression and distribution of sodium channels during development and maturation of the neuromuscular junction. J Neurosci 1993, 13:1326-1336.

53.

Sharp AA, Caldwell JH: Aggregation of sodium induced by a postnatally upregulated isoform J Neurosci 1998, 16:6775-8783.

54.

Hoch W, Ferns M, Campanelli JT, Hall ZW, Scheller RH: Developmental regulation of highly active alternatively forms of agrin. Neuron 1993, 11:479-490.

34. .

35.

Yu XM, Hall ZW: The role of the cytoplasmic domains of individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells. J Neurosci 1994, 14~785795.

36.

Clu Z, Ape1 ED, Doherty CA, Hoffman PW, Merlie JP, Huganir RL: The svnaose-associated orotein raosvn regulates tvrosine phosphorylation of proteins colocsiiz;d atnicotinic acetylcholine receptor clusters. MO/ Ce// Neurosci 1996, 8:171184.

37.

Huganir RL: Regulation by serine and tyrosine 2871279-294.

of the nicotinic acetylcholine receptor protein kinases. Adv Exp Med Biol 1991,

38. .

Colledge M, Froehner SC: Tyrosine phosphorylation of nicotinic acetylcholine receptor mediates Grb2 binding. J Neurosci 1997, 17:5038-5045. The authors demonstrate an interaction between GrbP and the Torpedo AChR 6 subunit, suggesting a potential role for AChR phosphorylation in the initiation of the GrbP-mediated signalling cascade at the postsynaptic membrane. 39.

Si J, Luo Z, Mei L: Induction of acetylcholine receptor gene expression by ARIA requires activation of mitogen-activated protein kinase. J Biol Chem 1996, 271:19752-l 9759.

40.

Tansey MG, Ghu GC, Merlie JP: ARIAIHRG regulates AChR E subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway. J Cell Biol 1996, 134:465-476.

41.

Altiok N, Altiok S, Changeaux J-P: Heregulin-stimulated acetylcholine receptor gene expression in muscle: requirement for MAP kinase and evidence for a parallel inhibitory pathway independent of electrical activity. EMBO J 1997, 16:717-725.

42.

43.

44.

Sealock R, Wray BE, Froehner SC: Ultrastructural localization of the Mr43,OOO protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies. J Cell Biol 1984, 98:2239-2244. Froehner SC, Luetje CW, Scotland PB, Patrick J: The postsynaptic 43 K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron 5:403-410.

1990,

Phillips WD. Koota C. Blount P Gardner PD. Stenibach JH. Merli’e JP: ACh receptor-rich membrane domains organiied in fibroblasts by recombinant 43-kilodalton protein. Science 1991, 251:568-570.

channels of agrin.

spliced

55. .

Gee SH, Madhavan R, Levinson SR, Caldwell JH, Sealock R, Froehner SC: Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophinassociated proteins. J Neurosci 1998, 18:128-l 37. This paper demonstrates, along with [62-l, that the PDZ domain of syntrophins mediates an association with the carboxyl terminus of NaChs, thereby targeting them to the dystrophin complex in muscle. 56.

Adams ME, Dwyer TM, Dowler LL, White RA, Froehner SC: Mouse a l- and 6 2-syntrophin gene structure, chromosome localization, and homology with a discs large domain. J Biol Chem 1995,270:25859-25865.

57.

Peters MF, Adams ME, Froehner SC: Differential syntrophin pairs with the dystrophin complex. 138:81-93.

58.

Michalak M, Opas M: Functions of dystrophin and dystrophin associated proteins. Curr Opin Neural 1997, 10:436-442.

59.

Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M: Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 1995, 378:8588.

60.

Kornau HC, Schenker LT, Kennedy MB, Seeburg PH: Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 1995, 269:17371740.

61.

Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC: Recognition of unique carboxy-terminal motifs by distinct PDZ domains. Science 1997, 275:73-77.

association of J Cell Bioll997,

Schultz J, Hoffmuller U, Krause G, Ashurst J, Macias M, Schmieder P, Schneider-Mergener J, Oschkinat: Specific interactions between the syntrophin PDZ domain and voltagegated sodium channels. Nat Struct Biol 1998, 5:19-24. See annotation [55*]. 62. .

63.

Deconinck AE, Potter AC, Tinsley JM, Wood SJ, Vater R, Young C, Metzinger L, Vincent A, Slater C, Davies KE: Postsynaptic abnormalities at the neuromuscular junctions of utrophin deficient mice. J Cell Biol 1997, 136:883-894.

64.

Grady RM, Merlie JP, Sanes JR: Subtle neuromuscular defects utrophin-deficient mice. J Ce// Biol 1997, 136:871-882.

in

Signals mediating ion channel clustering at the neuromuscular junction Colledge

65. ..

Deconinck AE, Rafael JA, Skinner JA, Brown SC, Potter AC, Metzinger L, Watt DJ, Kickson JG. Tinsley JM, Davies KE: Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 1997, 90:717-727. See annotation [66*-l.

363

67.

Cohen NA, Kaufmann WE, Worley PF, Rupp F: Expression of agrin in the developing and adult rat brain. Neuroscience 1997, 76:581-596.

68.

Burns AL, Benson D, Howard MJ, Margiotta JF: Chick ciliary ganglion neurons contain transcripts coding for acetykholine receptor-associated protein at synapses (rapsyn). I Neurosci 1997, 17:5016-5026.

69.

Gorecki DC, Abdulrszzak H, Lukasiuk K, Barnard EA: Differential expression of syntrophins and analysis of alternatively spliced dystrophin transcripts in the mouse brain. fur J Neurosd 1997, 9:965-976.

70.

Lin JW, Wyszynski M, Madhavan R, Sealock R, Kim JU, Sheng M: Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NRl. J Neurosci 1998, 18:2017-2027.

66. ..

Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS, Sanes JR: Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 1997, 90:729-738. This paper, together with [65**], describes mutant mice lacking both dystrophin and utrophin. In contrast to the mild phenotypes of single mutants, the double mutants display severe dystrophic symptoms, closely resembling those seen in Duchenne muscular dystrophy. Thus, these mice should prove a useful model for studying the pathogenesis of the disease, as well as testing therapeutic strategies. The unexpected result is the subtle phenotype of the neuromuscular junctions, which are nearly normal.

and Froehner