The basement membrane at the neuromuscular junction: a synaptic mediatrix

The basement membrane at the neuromuscular junction: a synaptic mediatrix Salvatore Carbonetto and Michael Lindenbaum McGill University, Montreal, Canada The basement membrane at the neuromuscular junction directs formation of pre- and postsynaptic elements at this synapse. Efforts to understand the molecular basis for development of the postsynaptic specialization have brought new insights into extracellular matrix proteins and their cell-surface receptors. Recent evidence for an agrin receptor and mice null for the s-laminin gene have reinforced the function of the basement membrane in both orthograde and retrograde signalling across the synapse. Current Opinion in Neurobiology 1995, 5:596-605

Introduction The extracellular matrix (ECM) of skeletal muscle includes a basement membrane sleeve that is differentiated into distinct synaptic and extrasynaptic domains around each myofiber. The extrasynaptic region of this membrane is a structural component that interacts with dystrophin-associated membrane proteins at the surface to buttress the cell against the stress of repeated contractions [1]. Genetic defects in dystrophin, a subplasmalemmal cytoskeletal protein, in the dystrophin-associated protein adhalin, or in the laminin 0~2 chain, lead to muscular dystrophies with minor effects on the neuromuscular junction (NMJ). In addition to acting as a superstructure, the synaptic basement membrane controls the regeneration of both a mature nerve terminal and postsynaptic apparatus [2]. The ECM protein agrin stinmlates acetylcholine receptor (AChR) aggregation, one of the earliest detectable events in synapse formation. This ultimately leads to a mosaicism in the multinucleated myofibers that is so profound that the subsynaptic region behaves ill many respects like a cell within a cell, transynaptically organized by signals released from the nerve terminal that regulate the muscle at virtually all levels, from gene transcription to post-translational modification of proteins. The signalling is bidirectional, however, as nmscle influences development of the nerve. Ill skeletal muscle, the basement menlbrane seems to }lave been co-opted during evolution to serve as a stable template for synapse formation, possibly because once outside of the cell, it escapes the normal turnover of cellular proteins.

In this review, we discuss the function of the E C M in forming the NMJ, with special emphasis on the postsynaptic membrane and its high concentration of AChRs, the hallmark of an NMJ. Our mandate is to review advances made over the past year (see [3] for a comprehensive review), and we do this ill the context of a working model of NMJ formation that deals with the earliest events in synapse formation. Recent progress has made it obvious that this reductionist approach is paying dividends, not only in our understanding of AChR. aggregation and the regulation of other postsynaptic molecules, but also of the difl'erentiation of the nerve terminal.

Specialization of the synaptic basement membrane Before innervation, AChP,.s and acetylcholinesterase (ACHE) are distributed uniformly over the muscle cell sur£ace. A rough scenario of NMJ formation would have the growth cone invading relatively undifferentiated nmscle masses in the embryo, releasing agrin onto the skeletal myotube. Agrin causes AChR.s within the plasma membrane to aggregate under the nerve terminal. The growth cone also releases acetylcholine (ACh), and a protosynapse forms within minutes of its contact with the muscle cell, triggering electrical activity that regulates expression of A C h R subunits [4] and AChE [5,6]. The motoneuron also releases neuregulins, a family of growth factors (including AChlZ-inducing activity

Abbreviations

ACh--acetylcholine; AChE--acetylcholinesterase; AChR--ACh receptor; ARIA--AChR-inducing activity; bFGF basic FGF; CGRP--calcitonin gene related peptide; DGC~ystrophin-associated glycoprotein complex; DRP~dystrophin-related protein; ECM-~extracellular matrix; EGF---epidermal growth factor; FGF--fibroblast growth factor; G domain--globular domain; HB-GAM--heparin-binding growth-associated molecule; HSPG--heparan sulphate proteoglycan; mAb--monoclonal antibody; NCAM--neural cell adhesion molecule; NMJ--neuromuscular junction; NOS--nitric oxide synthetase. 596

© Current Biology Ltd ISSN 0959-4388

The basement membrane at the neuromuscular junction Carbonetto and Lindenbaum

[ARIA], glial growth factor [GGF], Neu-differentiation factor [NDF], and heregulin) that become incorporated into the synaptic E C M ([7]; see Carraway and Burden, this isstie, pp 606-612). Neuregulins bind to epidermal growth factor (EGF)-like receptors (erbB2/B3) in the postsynaptic menrbrane, activating their endogenous tyrosine kinase activity, to stinmlate synthesis of AChK [7,8]. Neuregulin, possibly acting in conjunction with calc!Ltonm gene related peptide (CGRP) [9], contributes to the localization o f AChP,.s at the fornring NMJ by stinmlating transcription of AChR subunits in the subsynaptic nucleus [6,7] and by helping to maintain their: numbers in the ~lce of decreased synthesis of AChRs over the entire myofiber caused by muscle activity. Electron microscopy has shown that a detectable basement membrane does not appear within the synaptic cleft until shortly after NMJs have begun to form, suggesting that agrin may be an early player in the coalescence of a synaptic basement membrane. Moreover, once assembled, the basement membrane at the synapse is indistinguishable ultrastructurally from that over the rest of the cell. Yet, relatively subtle differences in composition can have a profound effect on basement menrbrane function with little, change in ultrastructure. For example, in mice null lbr the laminin [32 gene [10"], kidney glomerular basement membranes appear normal, possibly due to compensatory upregulation of the [31 chain, but filtration in the kidney is disrupted and these mice develop glomerular nephritis. Like all basement membranes, the junctional membrane contains laminin, collagen IV, entactin and heparan sulfate proteoglycan (HSPG), although they are present as distinct synaptic isoforms in each instance. Hall and Sanes [3] have enumerated these molecules concentrated at the NMJ. An update o f ECM components on this list should include a new form of entactin, an old protein involved in basement membrane assembly 111], and ot-dystroglycan, a dystrophin associated protein (Fig. 1; A Leschziner, SH Gee, M Lindenbaum, S Carbonetto, unpublished data). An emerging idea is that proteins are dit-ferentially glycosylated m thc subsynaptic region and that this process is under neural control and may have functional consequences. Such differences are apparently reflected in tile very structure o f tire Golgi apparatus, which is more prominent under the synapse than around any of tire extrajunctional nuclei [12 I. Following denervation, there is re expression of the (;olgi around extrajunctional nuclei, approximating the distribution seen before innervation. One likely targt't of neurallv conm)lled glycosylation is cntactin, which has a z~ovel carbohydratc epitopc at the synapse [111. Another potential target of diffe:ential glycosylation is c~-dystroglycan, which is a mutinous peripheral membrane glycoprotcm [13] and part of a complex o f at least seven surface proteins linked to dystrophin 11[ (Fig. 1). 0t-Dystroglycan and othe: dystrophin-associated proteins (discussed below)

are expressed diffusely on the muscle surface, but are concentrated at NMJs. Dystroglycan is apparently synthesized as a 97 kDa precursor polypeptide in rabbit muscle, which is cleaved to give rise to amino-terminally derived ot-dystroglycan, with a predicted molecular weight o f - 5 5 k D a , and carboxw-terminally derived [3-dystroglycan, a transmenrbrane protein linked to 0t-dystroglycan and part of the dystrophin-associated glycoprotem complex (DGC) [1]. 0t-Dystroglycan is heavily glycosylated and increases in size in developing chick nmscle from <120kDa before innervation to over 200 kDa in lnature muscle. Denervation of adult muscle leads to a decrease in the size of ct-dystroglycan, again, due to post-translational modification, most likely glycosylation (A Leschziner, 8H Gee, M Lindenbaunr, S Carbonetto, unpublished data). More direct evidence that carbohydrates may be involved in synapse formation comes from studies with the plant lectin VVAB4 [14°], which selectively labels NMJs by binding to X-acetyl-galactosamine, a relatively rare carbohydrate in glycoproteins. VVAB4 stimulates aggregation of AChP,,s on myotubes in culture and potentiates tile effect of agrin. More interestingly, ~\Vacetyl-galactosatnine bound to a carrier protein inhibits agrin-stimulated aggregation. Neither functional agrin nor the muscle form of 0t--dystroglycan, an agrin-binding protein (discussed below), have been shown to bind VVAB4 ([14"]; cf. [13]), so the players in this hypothetical, lectin-Iike interaction have yet to be identified.

Laminins

Laminins are heterotrmmrs consisting of oc (formerly A), [3 (formerly B1) and T (formerly B2) chains. There are at least three or four 0:, four [~ and two y chains that assemble to form seven to nine known heterotrmlers. Laminins containing tile [32 chain (formerly s-laminin) are localized to the NMJ and kidney glomeruhls. Sanes and co-workers (PT Martin, AJ Ettinger, JP,, Sanes, personal communication) have generated chimeric molecules to cvaluate targeting o( [~2-containing laminins to the NMJ. They have found that laminins with chimeric [~,1/[~2 chains are localized at spontaneously formed AChP- aggregates on cultured myotubes and that a 16 amino acid sequence m the carboxyl termitms (domain i) of the [~1 chain is necessary for targeting. There are no consensus sequences for .\'- or O-linked glycosylation in this l(,mer, so carbohydrates seem not to be as important in this targeting as they arc in other cells [151. Contained within the l(,iner, however, is tire tripcptidc LIKE (Leu-Arg-Glx), which has been previously proposed as a stop sib~al for growth cones. It is ~ir to say that this intriguing proposition has not been accepted with uniform enthusiasm among ECM biologists, as individual laminin chains seem to have

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Fig. 1. A diagram of the NMJ, including (:omponents of the junctional basement membrane (BM), receptors in the plasma membrane and subplasmalemmal cytoskeletal proteins. Illustrated from left to right are laminin containing [~2, ~,1 or (,2 (hains interacting with a spe(ially glycosylated form of entactin (S-entactin) localized to the junction, c~1 and (~2 chains of laminin bind to {~-dystroglycan ((t-DG) a peripheral membrane protein and part of the DGC, which includes the transmembrane proteins [3-DG, adhalin (adh), 25 kDa and 35 kDa proteins, as well as two intracellular peripheral membrane proteins A 0 and cd/[~2 forms of syntrophin (Syn) that interact with dystrophin. The latter isoform of syntrophin is localized to the junction. Grb2 is associated with [3-DG and is an adapter molecule that (ontains an SH:~ (sr( homology ',) domain that binds to phosphotyrosylated/proline-rich protein domains. The junctional BM also contains ARIA, a neuregulin that activates erbB2/B3, a heterodimeric receptor tyrosine kinase of the EGF receptor family. AChRs are shown phosphorylated and aggregated via the 43 kDa protein (Rapsyn), which may associate via its zinc finger motif. AChE is shown as one of three asymmetric forms consisting of 12 globular subunits linked to a collagenous tail, HSPG, such as perlecan, is bound in the junction, as is HB-GAM, and both are shown in a hypothetical interaction at the junction. Agrin is also a HSPG and binds to (x-DG. The junctional BM also contains an isoform of type IV collagen, which is distinct from that in the extrajunctional BM. The extrajunctional BM includes more common isoforms of collagen IV, laminin and entactin, and its DGC is associated with dystrophin rather than DRP and syntrophin od only. Finally, the diagram depi(ts two instances of orthograde signalling (black arrows), namely release of CGRP and ARIA by the nerve impinging on the nu(leus, and one retrograde pathway (clear arrow) to illustrate ECM proteins targeted to the junctional cell surface and acting back on the nerve. little biological activity. However, Porter et al. [16"] report that purified fusion proteins containing LP,.E but not L R A (Leu-Arg-Ala) or Q R E (Gln-Arg-Glx) inhibit neurite outgrowth on laminin and fibronectin but not collagen IV. Growth cones contacting a border o f LP, E-containing fusion proteins fail to extend across, but grow instead along it without collapsing. T h e 16mer appears to contain a growth cone stop signal (i.e. LP,.E) within a larger sequence that targets it to its site of action on the cell surface - - an adnfirable degree of parsimony for any protein. John Merlie contributed importantly over many years to our understanding of the molecular nuts and bolts of synaptogenesis. N o w his laboratory has employed gene inactivation by homologous recombination in e m b r y -

onic stem cells to generate mice null in genes implicated in synapse formation (laminin 2, rapsyn/43 kl)a protein, agrm). Their first offering is a lovely demonstration that the laminin [~2 chain is necessary for NMJ development I17"']. Mice null for the [~2 chain have NMJs with dinfinished numbers of synaptic folds, a reduction in the levels of neural cell adhesion molecule (NCAM) normally found at the bottoms of the junctional folds, as well as some separation o f the nerve terminal fiom the muscle. On the presynaptic side. vesicles arc dispersed throughout the terminal rather than localized at release sites normally found apposed to the junctional folds. In addition, the frequency of spontaneous translnitter release in the lnutant is substantially lower than that observed in wild-type mice. Thus, the [~2 chain seems to

The basement membrane at the neuromuscular junction Carbonetto and Lindenbaum

be involved in both orthograde and retrograde signalling between nerve and muscle. As the [~2 chain is found at the NMJ as part of a laminin heterotrimer, one might reasonably expect that mutations in other laininin subtmits would have an overlapping phenotype. In fact, the dystrophic inouse (dy/d),) has a naturally occurring mutation in the (*2 gene resulting in aberrant expression of this chain [18]. In this mutant, synaptic folds are nfissing, the nerve terminal is separated from the inuscle and secretory vesicles are dispersed within the terlninals [19] --- all reminiscent of the [32 null phenotype. There is also an alteration in spontaneous, but not evoked, ACh release in the dy/dy lnutant, with a high incidence of large spontaneous synaptic potentials that have slow rise times [20]. Moreover, in both the [~2 null and dy/dy mouse mutants, Schwann cell processes are interposed within the synaptic cleft [17"',19]. It would be inost exciting iflaminin 4 (0t2, [~2, y1) was directly responsible for these eft"ects by aligning postsynaptic gutters with presynaptic release sites. Consistent with this scenario, in the [~2-chain knockout, synapsin-1 appears more easily removed from the membrane, possibly indicative of loss of cytoskeleton-membrane-ECM linkage [17"]. Alternatively, invasion o f Schwann cell processes into the synaptic cleft may cause some or all of these et]%cts [17"]. There is as yet no evidence of axonal overgrowth at the NMJ of these mice, as might be predicted with the loss of the [~2 motor neuron stop signal. The [32 chain, although sufficient to stop nerve growth in culture, may not be necessary i~z l,il~o (in £1ct, the sequence is mutated in the human ~2 chain) or possibly its deletion is compensated for by upregulation of other LRE-containing proteins at the synapse.

Agrin and other factors that aggregate AChRs Agrin is a multifunctional molecule with a core protein of approximately 200 kDa and a carboxyl terminus that binds heparin and mediates AChlL aggregation (Fig. 2). In 1995, two labs [21",22"] reported that agrin ifz sire is a HSPG. Primer extension analysis of what was thought to be a full length chick agrin eDNA revealed a 50bp extension at the 5' end encoding 15 amino acids highly homologous to a HSPG (AJ Denzer, M Gesemann, B Schumacher, MA 1Luegg, personal communication). A monoclonal antibody to agrin proteoglycan labels motor endplates as well as the developing retina and optic nerve [22°]. Apparentl',; in previous studies of agrin purified from Torpedo, the proteoglycan (>400 kDa) was excluded from gels during electrophoresis and went undetected in favor of smaller breakdown products that were bioassayed. These results raise questions concerning fimctions of agrin that may not have been revealed in studies with fragments of native agrin or 'flail-length' recombinant protein. AChlL aggregation ha,,; been mapped within the carboxyl ternmms, so it seems unlikely that the small

amino-terminal extension would alter this [21",23"]. On the other hand, agrin proteoglycan interacts with a well-characterized heparin-binding site on N C A M [22"] that can modulate homophillic binding. N C A M is found at the ECM of the developing NMJ [3] and is upregulated with denervation, raising the possibility that agrin may regulate the adhesiveness of the junctional region. Agrin expression has taken on greater complexity than previously thought. There is a splice site in the newly unidentified amino terminus consisting of a seven amino acid insert that is found in developing motoneurons but not in nmscle (Fig. 2; M Gesemann et al., personal communication). Within the second lanfinin repeat at the 3' end (site A in chick agrin, site Y in rat agrin), a four amino acid peptide may be added, and just 5' of the last laminin-like region, an 8mer, an 1 liner or both can be inserted (chick agrin site B, rat agrin site Z). The presence of the 8mer (or the 19mer, which contains the 8mer) at the B (Z) site is necessary for maximal aggregating activity [24]. Chick motoneurons contain all possible B inserts, but the B19 insert (A4 B19) is most abundant during the period of synapse formation [25]. Thus, neurons can express multiple agrin transcripts that change in amounts during development, but they synthesize the most active forms during NMJ formation [25,26]. Agrin is not the only factor known to stimulate AChlL aggregation, but it appears to be the most physiologically significant one [27]. For example, basic fibroblast growth factor (bFGF) stimulates AChlL aggregation [3], but it is not clear whether bFGF is synthesized in or released by motoneurons. Earlier work demonstrated that CG1LP increases AChlZ expression [9]. CGILP accumulates at motor nerve terminals subsequent to nerve crush, and electrical stimulation causes release of these stores 128[, which may be important for re-establishing NMJs after injury. Finally, a protein called heparin-binding growth-associated molecule (HB-GAM, pleiotrophin, P18) is found on the surface of cultured muscle cells, but is concentrated at spontaneous AChR, clusters or those induced by a variety of means, including polystyrene beads [29"]. HB-GAM adsorbed to polystyrene beads stimulates AChlL aggregation and this is blocked by addition of tyrosine kinase inhibitors, similar to agrin-induced clustering (discussed below).

Agrin receptors Estimates of the stoichiometry of agrin to AChlL (-1:50) suggest that AChlL aggregation via this large protein is amplified through a cell-surface receptor. Indeed, agrm stimulates phosphorylation of proteins in culture (discussed below). Four labs have found that cc-dystroglycan in muscle cells is a major agrin-bmdmg protein [ 3 0 " - 3 3 " ] . As noted above, cc-dystroglycan is

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Fig. 2. A schematic representation of agrin. The molecule can be roughly divided into two halves, an anqno-ternqnal portion necessary for insertion of the molecule into the basement membrane and a carboxy-terminal half encompassing the AChR clustering activity. Notable features of the carboxy-terminal agrin fragment include four EGF-like domains and three laminin G type repeats, the se(ond of whk h ha,, been implicated in binding to (,-dystroglycan. Also note the presence of three alternative splice sites in the carboxy terminal half of the rat molecule (two in chick). Inserts of 8 amino acids (aa) or 19 aa at the Z site (site B in chick) of neurally expressed agrin have been correlated with maximun/ levels of AChR clustering activity. A novel amino-tem~inal spli(e site has recently been identified but it's functional role has yet to be ascertained. An extreme carboxy-terminal 21 kDa agrin fragment (ontaining the Z (B) site as well as the third G domain possess(,s AChR clustering activity in the absen(e of (t dystrogly(an binding.

part of a complex of dystrophin-associated proteins (Fi X. 1) that has the potential to trap AChRs by linking them to dystrophin-related protein (DI~,P; also known as utrophin) within the cell. That o,-dystroglycan fimctions in aggregation is controversial and helps explain why these four papers have spawned an equal nuinber of reviews (see e.g. [34]) over the past year. ct-I)ystroglycan binds to the last two globular (G) domains at the end of the 0~1 or ct2 chain within the long arm of laminin [351. Agrin contains three repeats m its carboxyl terminus homologous to the (; domains m laminin (Fig. 2). In addition, muscle ~-dystroglycan had been suggested to be a protcoglycan (however, of. [13]), and several earlier observations have implicated HSPGs in AChB, aggregation (see e.g. [24]). With this background, three laboratories have Focused on (t-dystroglycan and have shown that it brads agrm. In all cases, these observations relied heavily on a monochmal antibody (mAb IIH(,) that specifically recognizes ot dystroglycan, at least in part via a carbohydrate epitope [36 I, and blocks agrin binding [31"--33"]. In a separate approach, Bowe ct ,ft. [3I)"1 at'finitv purified a single agrin-bmding protein from 7brpcdo electric organ that was identified by protein microsequencing as c~-dystroglycan. Is 0t dystroglycan a fimctional agrin receptor? The G domain of laminin contains a major heparin binding site, and binding of agrin to 0{,-dystroglycan is inhibited by heparin or laminin, as well as by removal or

calcium. Both hcparin and calcium chelators inhibit AChP,, clustering, whereas laminin is a weak ag{mist of clustering. In addition, in a muscle cell lmc deficient in glycosaminoglycan synthesis and poorly responsive to soluble agrin, 0t-dystroglycan binds agrin weakly [31"°]. These indirect observations are consistent with the notion that the G domains within agrin harbor a heparin-binding site in or near the aggregation-activat ing region, and that binding to this region is inhibited by mAb IIH(,. Gee ct al. [31"] and Campanclli ~'t al. [32 °'] have reported that mAb IIH6 inhibited or dispersed AChI~ aggregates stimulated by agrin in (:2 muscle cells. On the other hand, Sugiyama et al. [33"] saw m~ et~'ct of the same monochmal antibody on the same cell lines. Moreover, they reported the Bo and B8 isoforms or agrin (noted above) both bind equally to (x-dystroglycan, even though the B8 form is approximately l{l(l(M-old more active in stilnulating AChl~, aggregation. Extending these latter observations, (;escm;mn ~'r ,;/. [21 °] have identified a carboxy terminal fiaglnent (21 kl)a) of rccolnbinant agrm that stimulates maxi real ACht< aggregation, although at a much higher concentration than larger carboxy terminal t-ragmcnts. However, this (ragnlellt does llOt contain the sequence (Lys Scr Arg Lys) at site A that is necessary for hcparin binding (M (;eselnann
The basement membrane at the neuromuscular junction Carbonetto and Lindenbaurn

that sequences of agrin outside this region cooperate ill stimulating aggregation. On the face of it, these data argue that a-dystroglycan is not fimctional ill AChR aggregation. This conclusion, however, hinges on the lack of correlation between agrin binding to a.-dystroglycan and biological activity, which has several possible flaws. First, ill its native state ct-dystroglycan is found associated at the sarcolemma wkh other members o f the DGC. The ct-dystroglycan used in binding studies is either a partially denatured protein or a purified protein isolated from other members of the complex that may alter its binding properties. Second, the DGC itself varies in composition. For example, it appears to interact with DRP at crests of foMs within the NMJ, but interacts with dystrophin elsewhere. In addition, a particular form of syntrophin (132 syntrophin), all intracellular inember of the complex, is localized at the endplates [37[. As endplates represent only a very small area of the total myotube suttee, this junction-specific complex may represent only a small proportion of the total ill muscle extracts used in binding studies. Moreover, after all extensive search, 0:-dystroglycan was the only protein identified as all agrm-bmding protein ill Torpedo electric organ [30°']. One explanation to reconcile these conflicting observations is that a-dystroglycan is a co-receptor that increases the affinity, or avidity of the cell for agrin and its chances of interacting with the primary receptor [21",33°°]. There is precedent for this ill the instance of bFGE which binds to a proteoglycan co-receptor befi)re interacting with the FGF receptor [38]. Without interaction with ct-dystroglycan it may require very high concentrations of agrin to activate aggregation, hence the high concentration of the 21 kDa fragment required for nlaximal aggregation. Also, this is renmfiscent of observations that cell-bound, inuscle agrin (Bo) is almost as effective as B8-containmg agrm isoforms ill stimulatiug AChK clusters on C2, but not $27 cultured myotubes [39]. hmnobilizillg agrin at the surface of a cell may potentiate its effects. Regardless of its precise role as primary or co-receptor, C o h e n c t a l . [40 °] report that 0:-dystroglycan is positioned to function ill synaptogenesis. 0~-Dystroglycan is tightly co-localized with AChKs ill aggregates that form spontaneously on lnuscle cells. Both AChP,, and a-dystroglycan ill these aggregates disperse upon contact o f a cell by all innervating axon. Moreover, ot-dystroglycan is found co-localized with neural agrm and AChKs at the earliest detectable moments of synapse formation (<1 h). mAb IIH6 disrupts spontaneously formed, but not neurally induced, aggregates of A(]hP,. The lack o f effect on the Latter may reflect a large amount of agrin deposited by the neurite on the muscle and the relatively low affinity of this IgM monoclonal antibody. In anv case, redistribution of spontaneously tbrmed aggregates of 0t-dystroglycan along with A(ThP, s by the distant nerve suggests that both are at}-ected by intracellular signalling.

I11 a related study, Apel et al. [41] expressed dystroglycan along with AChK subunits and rapsyn in a heterologous cell type, the quail fibroblast lille QT-6. iR,apsyn, a 43 kDa protein, is found exclusively at the NMJ, where it associates with AChR in a 1:1 ratio. When expressed by itself in Q T - 6 cells, rapsyn was found to be distributed m clusters and could also induce AChl< clusters in an agrin-mdependent fashion when co-expressed with AChlK subunits ill the salne lille. Apel et al. [41] showed that dystroglycan co-localizes with rapsyn-mduced clusters both ill the presence and absence of co-expressed AChK subunits, whereas numerous control proteins fail to co-localize with rapsyn-mduced clusters. Dystroglycan expressed alone or in combination with AChR subunits is found distributed unifornfly ill the cell and does not itself induce AChR. clusters ill Q T - 6 cells. Finally the rapsyn-induced clusters do not appear to contain associated utrophin/dystrophin as assessed by mmmnocytochelnistr~; suggesting that these clusters represent all early stage ill the process of aggregation (see below for discussion of possible inechanisms of receptor clustering). These results provide further evidence that dystroglycan specifically interacts with rapsyn, probably through the [~ subunit, and may play a structural as well as catalytic role in the formation of AChlK clusters. Intriguingly; [3-dystroglycan, with which 0t-dystroglycan is tightly associated [30"], and syntrophins have several potential sites for tyrosine phosphorylation [42,43,44"], all indication that these moieties may be involved ill intracellular signalling and/or that phosphorylation may ill some way modulate the binding properties of these polypeptides. Yang et a/. [44"] report that Grb2 is found m cells associated with [3-dystroglycan. Grb2 helps mediate signalling via GTP-binding proteins by docking with phosphotyrosylated or prolme-rich proteins through its src holnologT domains. Grb2 is known to function ill ECM signalling through the integrin family of ECM/adhesive proteins, which may be a point of signal convergence between integrins and the DGC. A final twist ill this tale ofdystrophin-associated proteins follows from observations that c¢/~-dystroglycan is expressed ill the CNS and is found localized along with dystrophin and DKP within the synapse-rich, outer plexiform layer of the retina [451, as well as within the dendritic arbors of cerebellar Purkmje cells [13]. These data implicate the DGC ill central synapse forlnation and may help explain the high incidence of mental deficiency in boys with 1)uchenne muscular dystrophy (see Note added mproot). The ligand tbr 0~-dystroglycan ill brain is a matter of additional speculation. Agrm and the laminin a2 chain are expressed ill the retina [46,47]. In addition, the neurexins, which contain G domains homologous to laminin and agrm, are a very large family of receptors found on nerve terminals [48]. The newly discovered protein neuroligin is a transmembrane protein that interacts with a subset ofneurexms ([3 neurexins), which are dif}'erentiated by inserts within their G domains [48]. It appears that G domains ill several families of proteins

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602

NeuronM and glial cell biology (such as agrin, laminin, perlecan, and neurexins) may be important in synaptogenesis o f the nervous systenl via their interaction with more than one family of receptors, neuroligin and potentially ct-dystroglycan.

Mechanism of AChR aggregation Aggregation of AChlks is a nmltistep process in which receptors in a freely diffusible pool on the cell surface become trapped within loci triggered by agrin. These events are well-delineated in co-cultures of nerve and muscle where the nerve serves as a point source of agrin (as well as other factors) and frst stinmlates the formation of small clusters of AChlK [40"], which co-localize with rapsyn. These nficroclusters eventually mature by coalescing into uniform plaques. Actually, the nerve releases enough agrin to form long continuous aggregates initially on muscle cells, as stripes of agrin deposited by neurites on culture substrates coated with ECM proteins induce stripes of AChR on the cell [49]. This may be a clue that the accumulation or stabilization of AChIKs into larger aggregates, similar to those at the NMJ, requires the assembly of ECM proteins at the muscle cell surface. Consistent with this, Bixby [50"] reports that collagen biosynthesis is necessary for nerve-induced AChlK aggregation. However, short-term treatment with 6-hydroxyproline, an inhibitor of collagen synthesis, does not inhibit aggregation, suggesting that some conlponents of the ECM participate at later stages of synaptogenesis, such as m accumulation/stabilization of aggregates. What happens after agrin binds? A likely internlediary is rapsyn, which, when overexpressed ectopically in cells, assembles into small aggregates that cause clustering of co-expressed AChFZs [51]. Presumably in muscle cells, some post-translational event like phosphorylation mimics the effect ofoverexpression of the 43 kDa protein by increasing its ability to aggregate, for example. In chick muscle cultures, 0~, 1~ and T AChlK subunits are phosphorylated in response to agrin treatment [52]; however, only the ~ and 8 subunits are phosphotyrosylated [53",54], nlimicking the pattern of AChP, subunit phosphorylation elicited by innervation in nerve nmscle co-cultures [54]. [3 subunit phosphorylation occurs within 1-2 h after agrin treatment and correlates with the appearance of AChI'Z nficroclusters as well as with decreased ease in extraction of AChlKs from the membrane with non-ionic detergents [55]. Addition of staurosporine, an inhibitor of tyrosine kinases, and to a lesser extent, serine/threonine kinases, results in the inhibition of agrin-induced clustering in chick myotubes and agrin-induced phosphorylation of the 13 AChlK subunit [53°]. Thus, tyrosine phosphorylation of the [3 AChP,. subunit may in some way regulate its binding to the cytoskeleton [53%56]. Although it seems clear that there is a requirement for phosphorylation in A C h R aggregation, it is controversial

whether phosphotyrosylation of the 1~ AChlK subunit is a step in the pathway. For example, in rats, unlike in chicks, the ~ AChlK subunit is only observed to undergo phosphorylation postnatally, well after the onset of NMJ formation [57]. More importantly, elimination of the cytoplasmic loop of the ~ subunit where tyrosine phosphorylation occurs does not impede the ability of rapsyn to stimulate AChlK aggregates in non-muscle cells [58°]. It remains to be tested whether agrin-induced clustering is silnilarly unimpaired in such mutant forms. Other potential substrates for agrin-induced phosphorylation include members of the I)(,C, such as [~-dystroglycan and syntrophin, which interact with dystrophin and DIKP [59,60"-62"]. Phosphorylation in the extended spectrin repeat region of dystrophin by protein kinase A results in an increase in association of dystrophin with F-actin, whereas phosphorylation of sites in the amino-ternfinal 0t-actinin-like domain by protein kinase C results in decreased association with F-actin [63]. DP,.P may undergo similar modifications, suggesting another possible lnechanism for regulation of AChR aggregation. In suinmary, the transmembrane signal elicited by agrin may alter associations between components of the membrane-associated cytoskeleton, cytoplasmic proteins such as rapsyn, the AChP,. itself, and other as yet unidentiffed components. The altered binding characteristics of the cytoskeleton as well as the presence of bound neural agrin at the cell surface might then serve as a 'trap' to sequester AChRs that diffuse into those regions of the cell. One attractive hypothesis is that agrin-induced phosphorylation may modify the binding of 1)(;C COlnponents (such as ~-dystroglycan or syntrophin) to the dystrophin/DP, P (utrophin) cytoskeleton, which ula}, in turn, serve to tether the nascent clusters. The finding that rapsyn-induced (agrm-independent) microclusters are not associated with dystrophin/1)tKP (utrophin) could be evidence for such nascent c]ust¢:rs. Secondarily, the insertion of ECM components (e.g. laminin ~2 chain, muscle agrin, HSPG) into this region of microclusters may serve to consolidate further the nascent nficroclusters into the larger ones typically seen following agrin treatment or nerve contact. AChR clusters may form on the surface of myotubes spontaneously, but, in the absence of a synaptic ECM, they may remain relatively unstable. In this molccular view of the diffusion trap model, AChR aggregates are not only initiated by agrin, an ECM protein, but mature along with the assembly of a matrix at the cell surfi~ce.

Conclusions and prospects Gene-knockout technology applied to the NMJ through the et-forts of Merlie and co-workers 110",17"] have yielded insights into the function of lalninin [~2 chain in the genesis of presynaptic as well as postsynaptic structures. Mice null for rapsyn and agrin are eagerly

The basement membrane at the neuromuscularjunction Carbonetto and Lindenbaum 603 awaited, although it may be tbolhardy to expect that their phenotypes can be simply extrapolated from cell culture studies o f synapse formation, which are designed to minimize the multiplicity, of interactions that occur during development i~1 l,il,o. Identification of 0t-dystroglycan as a candidate receptor/ co-receptor in transmembrane signalling and ECM assembly has allowed us to form testable models for the mechanism ofagrin-induced clustering. Mapping of AChlq. clustering activity to carboxy-terminal regions of agrin should prove a valuable tool with which to identiff, possible novcl agrin receptors. Nevertheless, it lnay be too early to write off ct-dystroglycan and the dystrophin-associated cytoskeleton, especially m light of its localization at developing synapses and the potential of"~,-dystroglycan to participate in signalling pathways by virtue of interaction with Grb2. Experiments designed to perturb this interaction in muscle cell lines will providc a test of its involvement in clustering. The description of alternative inducers of AChR chtstering, such as the recently described effects of HB-GAM [291 , opens the possibility that nature may have evolved alte::native mechanisms of AChl~ aggregation. Research over the past year has also highlighted the later stages of AChB. aggregation, which appear to involve biosynthesis, targeting into the cell surface and incorporation of ECM proteins, such as laminin containing the [32 chain. In this regard, it ,,viii be important to have more detailed information of events related to coalescence of microclusters into large aggregates and their turnover at the cell surt~lce. Finally, with identification of erbB2/B3 as an ARIA receptor, modelers can plug in a major piece of-the puzzle on early NMJ formation. Research on this receptor tyrosine kinase will stimulate work on intracellular signalling triggercd by agrin as well. Involvement of AChP,. phosphorylation in this process should be resolved soon by inutational analysis ofAChP,, subunits. Then the hard work of describing the intermediates in the pathway will begin.

Acknowledgements We are gratefifl to our colleagues w h o have shared with us data that are m press or unpublished. We also thank M o n r o e C o h e n and Michael Ferns for their c o m m e n t s on this text. This work was supported by grants from the Muscular 1)ystrophy Association o f - A m e r i c a arm from the M I ( C Canada.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ,,. of outstanding interest 1.

Campbell KP: Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell 1995, 80:675-679.

2.

McMahan UJ: The agrin hypothesis. Cold Spring Harb Symp Quant Biol 1990, 55:407-418.

3

Hall ZW, Sanes IR: Synaptic structure and development: the neuromuscular junction. Neuron 199L 10:99-121.

4.

Gunderson K, Sanes JR, Merlie JP: Neural regulation of muscle

acetylcholine receptor ~- and ra.-subunit gene promoters in lransgenic mice. J Coil Biol 1995, 123:1535-1544. 5.

lasmin BJ, Lee RK, Rotundo RL: Compartmentalisation of acetylcholinesterase mRNA and enzyme at the vertebrate neuromuscular junction. Neuron 1993, 11:467-477.

6.

Grubic Z, Komel R, Walker WF, Miranda AF: Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcbolinesterase and its mRNA in cultures of human muscle. Neuron 1995, 14:317-327.

7.

Jo SA, Zhu X, Marchionni

8.

Chu GC, Moscoso LM, Sliwkowski MX, Merlie JP: Regulation of

MA, Burden SJ: Neuregulins are concentrated at nerve-muscle synapses and activate ACh-receptor gene expression. Nature 1994, 373:158-161.

the acetylcholine receptor subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation. Neuron 1995, 14:329-339. 9.

Fontaine B, Klarsfeld A, Hokfelt T, Changeux JP: Calcitonin gene related peptide present in spinal and motoneurones increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci Lett ]986, 71:59 65.

10. •

Noakes P(;, Miner JH, Gautam M, Cunningham JM, Sanes JR, Merlie JP: The renal glomerulus of mice lacking

S-laminin/laminin 2: nephrosis despite molecular compensation by laminin. Nature Genet 1995, in press.

Nole added in proof Brenman cl a/. [64] recently reported that neuronaltype nitric oxide synthetase (,NOS) is associated with dyst~rophin/DGC in skeletal muscle via a four amino acid sequence (Gly-Leu-Gly-Phe) in the amino-terminal domain of NOS. In the dystrophin mutant m d x mouse, as well as humans with Duchenne muscular dystrophy, NOS is tbund m the cytoplasm rather than the plasma melnbrane. Earlier studies had shown that NOS regulates contractiliw and synaptic transmission in skeletal muscle.

In analysis of the phenotype of the S-laminin targeted mice (see also [16*]), the authors noted an abnormality in the kidney glomeruli resulting in severe proteinuria, possibly the primary cause of death in these animals. Interestingly, little if any apparent defect in the glomeruli basement membrane was noted at the electron microscopic level. 11.

Chiu AY, Ko J: A novel epitope of entactin is present at the mammalian neuromuscular junction. J Neurosci 1994, 14:2809 2817.

12.

Jasmin BJ, Antony C, Changeux JP, Cartaud J: Nerve-dependent

plasticity of the Golgi complex in skeletal muscle fibres: compartmentalization within the subneural sarcoplasm. Eur J Neurosci 1995, 7:470-479.

13.

Smalheiser NR, Kim E: Purification

of cranin, a laminin binding membrane protein. Identity with dystroglycan and reassessment of its carbohydrate moieties. J Biol Chem 1995, 270:15425-15433.

604

Neuronal and glial cell biology 14.

*

Martin PT, Sanes JR: Role for a synapse-specific carbohydrate in agrin-induced clustering of acetylcholine receptors. Neuron

1995, 14:743-754. In previous reports, N-acetylgalactosamine glycoconjugates were found concentrated at the NMI. This study presents evidence that they function in AChR clustering. 15.

Fiedler K, Simon K: The role of N-glycans in the secretory pathway. Cell 1995, 81:309-312.

16. Porter BE, Weis J, Sanes JR: A mot,neuron-selective stop signal • in the synaptic protein s-laminin. Neuron 1995, 14:549-559. The authors have identified a 16 amino acid sequence within domain 1 of the laminin [32 chain, which is responsible for targeting the expression of this species to the NM]. This sequence contains a previously identified tripeptide that serves as a stop signal for axonal growth cones in vitro. 17.

Noakes PG, Gautam M, Mudd J, Sanes JR, Medie JP: Aberrant

*o

differentiation of neuromuscular junctions in mice lacking

s-laminin/laminin. Nature 1995, 374:258-262. Mice lacking the laminin [32 chain appear normal at birth but gradually weaken and die after about 2-4 weeks. Innervation is normal in terms of number and position of endplates, but the animals have several pre- and postsynaptic abnormalities. 18.

Xu H, Wu X-R, Wewer UM, Engvall E: Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama) gene. Nature Genet 1994, 8:297-301.

19.

Banker BQ, Hirst NS, Chester CS, Fok RY: Histometric and electron cytochemical study of muscle in dystrophic mouse. Ann NY Acad Sci 1979, 317:115-131.

20.

Carbonetto S: Neuromuscular transmission in dystrophic mice. J Neurophysiol 1977, 40:836-843.

21.

Gesemann M, Denzer AJ, Ruegg MA: Acetylcholine receptor aggregating activity of agrin is,forms and mapping of the active site. J Cell Biol 1995, 128:625-636.

•

A 21 kDa peptide consisting of the extreme carboxy-terminal of chick agrin, the B alternative splice site (with the 8 amino acid insert) and the third laminin-like G domain is sufficient to cause clustering of AChRs. Unlike full-length agrin, heparin does not block the clustering activity of this fragmenL These results suggest agrin uses the third G domain to stimulate clustering and that this site is separate from that recognized by o:-dystroglycan, 22. Tsen G, Halfter W, Kroger S, Cole GJ: Agrin is a heparan sulfate • proteoglycan. / Biol Chem 1995, 270:3392-3399. Agrin is secreted as a >400 kDa HSPG. Antibodies to this form immunohistochemically label regions of the brain as well as the NMJ, consistent with this representing an active form of agrin.

GAM) in the postsynaptic induction in cultured muscle cells. J Neurosci 1995, 15:3027-3038. HB-GAM is a muscle-derived growth factor, potent in clustering AChRs when h-nmobilized on polystyrene beads but relatively inactive in solution when added to cultured muscle cells.

30.

**

A protein complex from Torpedo electric organ purified on the basis of its high affinity for agrin is the Torpedo homologue of cd[~-dystroglycan. 31. •~

Campanelli JT, Roberds ST, Campbell KP, Scheller RH: Role

for dystrophin-assoclated glycoproteins and utrophin in agrininduced AChR clustering. Cell ]994, 77:663-674.

Gee et al. [31"'] and Campanelli et al. [32 •" ] both made use of rnonoclonal antibody IIH6 to c(-dystroglycan showing that it blocks specific binding of agrin to cc-dystroglycan. In addition, Gee et al. [31 *•] showed that this antibody inhibits agrin-induced AChR clustering in a dose-dependent manner, whereas Campanelli et al. [32•el noted that the antibody causes appearance of microclusters. Gee et al. [31 e.] showed that in a muscle cell line deficient in glycosaminoglycan synthesis and with impaired agrin-induced clustering, 0~-dystroglycan expression is greatly reduced when compared with wildtype cells. These data are consistent with a role for 0~-dystroglycan in agrin-induced clustering. 33. Sugiyama J, Bowen DC, Hall ZW: Dystroglycan binds nerve •• and muscle agrin. Neuron 1994, 13:103-115. This article raises the possibility that o.-dystroglycan is an agrin-binding protein but may not be a functional agrin receptor. 34.

Fallon JR, Hall ZW: Building synapses: agrin and dystroglycan stick together. Trends Neurosci 1994, 17:469-473.

35.

Gee SH, Blacher RW, Douville P), Provost PR, Yurchenco PD, Carbonetto S: Laminin-binding protein LBP-120 from brain

is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin. J Biol Chenr 199~, 268:14972-14980. 36.

complex as a transmembrane linker between laminin and actin. 37.

Smith MA, O'Dowd DK: Cell specific regulation of agrin mRNA splicing in the chick ciliary ganglion. Neuron 1994, 12:795-804.

26.

Ma E, Morgan R, Godfrey EW: Agrin mRNA variants are differentially regulated in developing chick embryo spinal cord and sensory ganglia. J Neurobiol 1995, 26:585-597.

27.

Cohen MW, Godfrey EW: Early appearance and neuronal

contribution of agrin like molecules at embryonic nerve-muscle synapses formed in culture. J Neurosci 1992, 12:2982-2992. 28.

Kramercy

NR,

Sealock

R, Froehner SC: [32

Aviezer [9, Hecht D, Safran M, Eisinger M, David G, Yayon A:

Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 1994, 79:1005-1013.

Ferns M, Hoch W, Campanelli JT, Rupp F, Hall ZW, Scheller RH: RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity in cultured my,tubes. Neuron 1992, 8:1079-1086.

40. •

Cohen MW, Jacobson C, Godfrey EW, Campbell KP, Carbonetto S: Distribution of (~-dystroglycan during embryonic nerve-muscle synaptogenesis. J Cell Biol 1995, 129:1093-1101. In co-cultures of Xenopus embryonic neurons and muscle, o. dystroglycan is detected at the earliest stages of the formation of synapses. Mab IIH6 application results in the break-up of spontaneous clusters into aggregates of microclusters but has little effect on nerve-induced clusters. These results suggest that 0:-dystroglycan may play an role in the early stages of synapse formation. 41.

Apel ED, Roberds SL, Campbell KP, Merlie JP: Rapsyn may

function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.

Sala C, Andreose JS, Fumagalli G, Lomo T: Calcitonin gene-

related peptide: possible role in formation and maintenance of neuromuscular junctions. J Neurosci 1995, 15:520 528.

Peters MF,

syntrophln: Iocalisation at the neuromuscular junction in skeletal muscle. Neuroreport 1994, 5:1577 1580.

Ferns M, Campanelli JT, Hoch W, Scheller RH, Hall ZW: The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans. Neuron 1993,

25.

Ervasti JM, Campbell KP: A role for the dystrophin glycoprotein / Cell Biol 1993, 122:809-823.

39.

11:491-502.

Carbonetto S:

32.

38.

24.

F, Lindenbaum MH,

Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell 1994, 77:675-686.

*',

Several regions of agrin required for AChR clustering are identified by deletion analysis or inhibitory monoclonal antibodies. These include the third EGF repeat, the second laminin like G domain as well as the extreme carboxy-terminal domain.

•

Gee SH, Montanaro

See annotation [32°°].

Hoch W, Campanelli JT, Harrison S, Scheller RH: Structural domains of agrin required for clustering of nicotinic acetylcholine receptors. EMBO J 1994, 13:2814-2821.

23.

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, 12:1173-11 80.

Neuron 1995, 15:115-126.

42.

Wagner KR, Huganir RL: Tyrosine and serine phosphorylation

29.

Peng HB, Afshan All A, Dai Z, Daggett DF, Raulo E, Rauvala H:

of dystrophin and the 58 kDa protein in the post-synaptic membrane of the Torpedo electric organ. J l',!eurochem 1994,

•

The role of heparin-binding growth-associated molecule (HB-

62:1947 1952.

The basement m e m b r a n e at the neuromuscular j u n c t i o n C a r b o n e t t o and L i n d e n b a u m 43.

Yamamoto H, Hagiwara Y, Mizuno Y, Yoshida M, Ozawa E:

Heterogeneity of dystrophin associated proteins. J Biochem

56.

choline receptors with the cytoskeleton by agrin-activated

(Tokyo) 1994, 114:132-139.

44.

Yang B, Jung D, Motto D, Meyer J, Koretzky G, Campbell KP:

•

SH3 domain-mediated interaction of dystroglycan and Grb2.

J Biol Chem 1995, 270:1-4. This paper is the first demonstration of a member of the dystrophin complex interacting with a protein, Grb2, implicated in signal transduction.

45.

Montanaro F, Carbonetto S, Campbell KP, Lindenbaum MH: Dystroglycan expression in the wild type and MDX

mouse neural retina: synaptic colocalization with dystrophin, dystrophin-related protein but not laminin. J Neurosci Res 1995, in press. 46.

Morissette N, Carbonetto S: Merosin ct2 chain is found within the pathway of avian and routine retinal projections. J Neurosci 1995, in press.

47.

Ma E, Morgan R, Godfrey EW: Distribution of agrin mRNAs in the chick nervous system. J Neurosci 1994, 14:2943-2952.

48.

Ichtchenko K, Hata Y, Nguyen T, Ulrich B, Missler M, Moomaw C, Sudhof TC: Neuroligin 1: a splice site-specific ligand for [3-neurexins. Cell 1995, 81:435-443.

49.

50.

•

Cohen MW, Moody-Corbett F, Godfrey EW: Former neuritic

57.

Qu Z, Moritz E, Huganir RL: Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction. Neuron 1990, 2:367-378.

58.

Yu X-M, Hall ZW: The role of cyloplasmic domains of

•

individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells. J Neurosci 1994, 14:785-795.

Deletion of the region of the AChR [3 subunit encompassing the agrin-sensitive tyrosine phosphorylation site does not impede 43 kDa induced AChR aggregation in a non-muscle cell line, suggesting that tyrosine phosphorylation of this subunit is not required for clustering. 59.

Suzuki A, Yoshida M, Hayashi K, Mizuno Y, Hagiwara Y,

Ozawa E: Molecular organization at the glycoprotein complex binding site of dystrophin: three dystrophin associated proteins bind directly to the carboxy-terminal portion of dystrophin. Eur J Biol 1993, 220:283-292.

60. Ahn AH, Kunkel LM: Synlrophin binds to an alternatively • spliced exon of dystrophin. J Cell Biol 1995, 128:363-371. See annotation [62"]. Suzuki A, Yoshida M, Ozawa E: Mammalian ctl and 131 syntrophin bind to the alternative splice-prone region of lhe dystrophin COOH terminus. J Cell Biol 1995, 128:373-381.

61.

Bixby JL: Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro. J

See annotation [62"].

Prolonged treatment with 6-hydroxyproline, an inhibitor of collagen synthesis, inhibits AChR aggregation in nerve muscle co-cultures implicating ECM synthesis in relatively late events in synapse formation. 51.

Froehner SC, Luetje CW, Scotland PB, Patrick J: The post-synaptic 43 kDa prolein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron 1990,

52.

Wallace BG, Qu z, Huganir RL: Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron 1991, 6:869-604.

5:403-410.

53. •

Wallace BG: Staurosporine inhibits argin-induced acetylcholine receptor phosphorylation and aggregation. J Cell Biol 1994, 125:661-668. Inhibition of agrin-induced clustering by an inhibitor of tyrosine phosphorylation implicates [3-AChR subunit tyrosine phosphorylation in the mechanism of aggregation. Qu Z, Huganir RL: Comparison of innervation and agrin-in-

duced tyrosine phosphorylation of the nicotinic acetylcholine receptor. J Neurosci 1994, 142:6834-6838. 55.

protein tyrosine kinase. J Cell Biol 1995, 128:1121-1129.

pathways containing endogenous neural agrin have high synaptogenic activity. Dev Biol 1995, 167:458-468.

Neurobiol 1994, 26:262-272.

54.

Wallace BG: Regulation of the interaction of nicotinic acetyl-

Wallace BG: Mechanism of agrin induced acetylcholine receptor aggregation. J Neurobiol 1992, 23:592-604.

•

62.

Yang B, lung D, Rafael JA, Chamberlain JS, Campbell KP: Identification of syntrophin binding to syntrophin triplet, dyslrophin, and utrophin. J Biol Chem 1995, 270:4975-4978. The four papers [59,60"-62"] describe binding in vitro to the carboxyterminal domain of dystrophin of [8-dysl:roglycan and the syntrophins. [8-dystroglycan binds within a cysteine-rich domain, whereas the syntrophins bind to a region within exons 73 and 74. Ahn et al. [60"] additionally show that the syntrophins also bind to utrophin and an 87 kDa postsynaptic protein, whereas Yang et al. [62"] demonstrate that the syntrophins self-associate. •

63.

Senter L, Ceoldo S, Meznaric

Petrusa M,

Salviati

G:

Phosphorylation of dystrophin: effects on actin binding. Biochem Biophys Res Comm 1995, 206:57-63.

64.

Brenman JE, Chao DS, Xia H, AIdope K, Bredt DS: Nitric

oxide synthetase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. ('ell 1995, 82:743-752, S Carbonetto and M Lindenbaum, Centre for Neuroscience P.esearch, McGill University', Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal P Q H3G 1A4, Canada. S Carbonetto e-mail: [email protected]

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