Agrin and the organization of the neuromuscular junction

Agrin and the organization Fabio Rupp, Werner

of the neuromuscular

Hoch, James T. Campanelli,

junction

Thane Kreiner and

Richard H. Scheller Howard

Hughes Medical

Institute,

Stanford

University,

California,

USA

Agrin is a component of the synaptic extracellular matrix and may regulate the organization of acetylcholine receptors and other synaptic molecules in both synapse regeneration and development. Analyses of cDNAs encoding agrin define a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and epidermal growth factor repeats.

Current Opinion in Neurobiology 1992, 2:88-93

Introduction Synapses mediate communication between neurons and other cells. An understanding of synapse formation during development and synaptic reorganization during learning is a critical issue in cellular neuroscience. The neuromuscular junction (NMJ), the synaptic connection between motor neurons and muscle fibers, has been widely studied in investigations of the function, development, and regeneration of synapses. The NMJ presynaptic active zone comprises plasma membrane specializations and accumulations of secretory vesicles that contain high concentrations of neurotransmitters. The intervening synaptic cleft is occupied by a basal lamina, which is highly enriched in molecules such as acetylcholinesterase [l], heparan sulfate proteoglycan [2], and s-laminin [3,4]. The postsynaptic membrane, just opposite the active zone, contains high concentrations of chemoreceptors strategically situated to detect secreted neurotransmitter. In this review, we focus on the molecules that are proposed to regulate the formation of the highly specialized NMJ.

Regulation

of acetylcholine

receptors

Regulation of the synthesis and accumulation of acetylcholine receptors (AChRs) at synaptic sites is one of the most intensely studied aspects of NMJ development [ 5,6]. Prior to innervation, AChRs are rather evenly distributed along muscle fiber surfaces. As axon terminals contact muscle fibers, receptors aggregate into high density patches at synaptic sites. Nuclei also accumulate at these synaptic sites in muscles; they express synaptic molecules, Including the AChR, whereas other nuclei in the muscle cell do not. The ability to culture muscle fibers has aided the development of in vib-o assays for studying receptor synthesis and localization [ 71. Several molecules

which are likely to play roles in regulating AChRs have been characterized. Two of these, the neuropeptide calcitonin gene-related peptide [8-lo], and the AChR inducing activity (ARIA) peptide [ 11,12*], regulate components of receptor synthesis. AChR clustering activity also appears to be mediated by multiple factors. When latex beads are coated with basic fibroblast growth factor (bFGF) and placed in contact with Xenopus muscle fibers, AChRs accumulate at contact sites [13*]. In contrast, when the beads are coated with bovine serum albumin, tenascin, or laminin, receptor clustering is not observed. This clustering effect is blocked by inhibitors of tyrosine kinases, and bFGF is inactive if applied to the muscle culture media in a bath [ 13.1.Taken together, these results suggest that receptor clustering activity is dependent on the context of bFGF presentation.

The involvement

of extracellular

matrix

components ~grin is a component of the synaptic basal lamina that is thought to be important in regulating NMJ organization [14]. Its discovery stemmed from an assay in which extracts of the electric organ of marine rays, enriched in extracellular matrix, were added to chicken muscle fibers in culture, whereupon AChRs clustered into patches [ 151.This assay was used to screen antibodies generated against extracellular matrix components, and resulted in a set of monoclonal antibodies which removed AChR aggregating activity from the extracts [16]. Histochemical studies using these antibodies demonstrate that lmmunoreactivity is localized to the synaptic basal lamina, and is also present in structures resembling Golgi cistemae in motor neuron somata [ 171. When nerves are ligated, immunoreactive material and AChR aggregating activity accumulate on the cell-body

Abbreviations AChR-acetylcholine receptor; ARIA-acetylcholine receptor inducing activity; bFCF-basic fibroblast growth factor; EGF--epidermal growth factor; NMJ-neuromuscular junction; PST-l--pancreatic serine trypsin inhibitor type I.

BB

@ Current Biology

Ltd ISSN 0959-4388

The organization of the neuromuscular junction Rupp, Hoch, Campanelli, Kreiner and Scheller

side of the ligature [ 181. Agrin immunoreactivity is also found on muscle fibers prior to innervation, and on fibers in culture, suggesting that agrin-like proteins are synthesized in muscle as well as nerve. Following nerve and muscle damage, agrin immunoreactivity remains associated with the synaptic basal lamina, suggesting that the molecule may function in synapse regeneration as well as synapse development [I4].

Agrin domain

structure

and expression

The monoclonal antibodies described above have been used to purify four proteins of 15OkD, 135 kD, 95 kD, and 70 kD. The 150 kD and 95 kD species are active in aggregating AChFs when applied in a bath to chick muscle fibers in culture [ 191. Furthermore, the amino-terminal amino acid sequences of the 150 kD and I35 kD proteins are identical to each other as are the aminoterminal amino acid sequences of the 95 kD and 70 kD proteins. To isolate cDNAs encoding agrin, a serum artibody was generated against the four protein species, and used to screen a cDNA expression library made with RNA isolated from electromotor neurons. The sequence of one of these clones predicts a protein that contains the amino-temrinal amino acid sequence of the 95 kD and 70 kD proteins. This clone presumably does not encode a fulllength protein as it does not contain the 150 kD and 135 kD amino-terminal sequences. To understand the nature of the agrin proteins in mammalian species, the ray sequence has been used to characterize a set of overlapping clones from rat embryonic spinal cord [20*]. Figure 1 shows a schematic diagram of the rat and ray proteins, derived from the analysis of recombinant cDNAs. The set of cDNAs from embryonic spinal cord deiine an open reading frame ilanked by inframe stop codons. The predicted initiator, Met, is followed by a hydrophobic stretch of amino acids with the characteristics of a signal sequence. This sequence probably directs the protein to the secretory pathway. tie Ieu-ArgGlu sequences are observed in the predicted rat protein, and may serve as neurite attachment sites [21]. Nine domains are homologous to the Kazal family of protease inhibitors [22], including pancreatic serine trypsin inhibitor type I (PST-I), submandibular trypsin inhibitors, and acrosin inhibitor I. The general organi zation of Cys residues in the Kazal protease inhibitor family is Cys-X&ysX7-CysX1eCys-X+ys-X&ys (where X is any amino acid) with the first and fourth, second and iifth, and third and sixth Cys residues disulfide linked, [23]. The protease inhibitors in the agtin protein contain 1623 amino acid extensions including three more Cys residues than the 56.amino-acid, PST-I. There are as yet no data concerning the function of this region of the agrin protein and, as a result, it is not known if these domains actually function as protease inhibitors. The disukide-linked motif present in the inhibitor domains may represent a structural feature with no relation to protease inhibitor function. It is intriguing, however, that sequence comparisons among a variety of distantly

related protease inhibitors reveal that the most highly conserved region is between the third and fourth Cys residues; this same region is also the most conserved among the agrin protease inhibitor domains. The interactions between proteases and their inhibitors have been studied in detail at the biochemical and structural levels. Protease inhibitors usually bind to their respective proteases with high affinity but present a peptide bond that is only slowly hydrolyzed [ 231. In PST-I, this bond is between Arg and Ile residues; this sequence is not conserved between different inhibitors, but is instead specific for particular proteases. The equivalent site in the agrin protease inhibitor domain is also not conserved between the different repeats. This suggests that either the repeats do not inhibit any known proteases, or the inhibitor func’ tion may be absent from these repeats. Located between protease inhibitor repeats 8 and 9 is a region of signiiicant homology ‘to domain III of the various subunits of laminin [ 241: The homology extends over 119 amino acids at a level of 39% identity, including 18 Cys residues. In the carboxy-terminal half of the protein, four epidermal growth factor (EGF) repeats are apparent [25]. In addition to the six Cys residues, the repeats are 25% identical at other amino acid positions. Further structural features include four potential N-linked glycosylation sites and two Ser and Thr rich domains, which may function as O-linked glycosylation domains in the agrin protein. Several forms of the agrin protein are generated by alternative RNA splicing. At position 1779, just prior to the second Ser/Thr rich domain, two alternatively spliced forms of the protein are produced by either including or excluding a nine amino acid sequence. This nine amino acid sequence includes two Thr residues and three Phe residues. A second alternatively spliced region is located toward the carboxyl terminus of the agrin protein, after the fourth EGF repeat. This region of the molecule can exist in four conformations generated by alternative splicing as follows: first, no additional exon sequences may be present; second, an eight amino acid sequence may be inserted at this site; third, 11 amino acids may be inserted at this site; and fourth, 19 amino acids, consisting of the eight plus the 11 amino acid sequences, may be inserted at this site. Thus, the combination of two RNA splicing alternatives at one site, and four RNA splicing alternatives at a second site, leads to a total of eight possible forms of the molecule in the rat. It is not yet known which of these alternatives exist in viva, as the variants have been derived from partial cDNAs and polymerase chain reaction analyses. A common method of defining important functional domains of proteins is to compare amino acid sequences between species. Figure 1 illustrates the percentage identity between the marine ray and rat agrin proteins in the particular structural domains discussed above. The relative positions of the Ieu-ArgGlu sequences and most of the N-linked glycosylation sites are not maintained. The two Ser/Thr rich domains are relatively poorly conserved in sequence, but their overall character is retained. Although the llanking protease inhibitor domains are relatively similar between rat and ray, the most highly

89

90

Development

Rat

DWFPTFFTC

Kazal protease inhibitor homolog

Signal sequence

I

I 1

2

3

4

\/

Laminin homolog LRE

5

6

7

BP

9 EGF repeat N-linked glycosylation 95 kD

LRE

200 amino acids

I

Ray

I

% Identity Imm

50 Cl

c25%_ identity

?? >25%

identity

~50%

identity

??>75%

identity

25

Fig. 1. A schematic illustration of the domain structure and evolutionary conservation of rat and marine ray agrins. The rat agrin is initiated with a hydrophobic domain that is predicted to function as a signal sequence. Nine regions homologous to Kazal protease inhibitors are indicated, one domain is similar to the laminins. Four epidermal growth factor (EGF) repeats are located in the carboxy-terminal portion of the protein. Leu-Arg-Clu (LRE) sequences are indicated, as are two domains rich in Ser and Thr (ST). Potential N-linked glycosylation sites are also shown. Alternatively spliced forms of the protein are produced by either including or excluding the amino acid sequences indicated above the rat protein. The amino-terminal sequence of the 95 kD protein active in receptor clustering is indicated by an arrow above the marine ray protein. The histogram illustrates the percentage identity between the marine ray and rat agrin proteins in the particular structural domains illustrated above.

conserved region is the laminin-homologous domain. In laminin, this domain promotes cell attachment, nidogen binding, and mitogenesis [ 241. Some or all of these functions may be present in agrin as well. The amino-terminal region of the 95 kD protein that is active in clustering receptors is indicated by an arrow in Fig.1. It is possible that this portion of the protein is sufhcient to induce receptor clustering. Of the four EGF repeats in this domain, the first is highly conserved with respect to the others and to flanking sequences. Other than this first repeat, there is no long stretch of outstanding amino acid identity between the two species in this part of the protein. Only a relatively short sequence may therefore be required for AChR aggregating activity, alternatively, the molecule may fold into a tertiaty structure that brings short conserved sequences in close proximity to generate a moiety active in receptor clustering. The availability of recombinant clones allows the investigation of sites of agrin gene expression using northern blotting and in situ hybridization. The gene is expressed most abundantly in embryonic brain and spinal cord, to a lesser extent in muscle, and below detectable levels in liver. In situ hybridization shows intense labeling of rat embtyonic day 15 motor neurons. At this stage of

development, the axons are first contacting muscle fibers, consistent with agrin playing a role in NMJ formation. These results are consistent with studies in the chick that demonstrate agrin immunoreactivity along muscle fibers prior to innervation [ 261. The immunoreactivity becomes progressively limited to synaptic sites as development proceeds. It remains to be seen if the alternatively spliced forms of the protein are differentially expressed in various tissues or at various times during development.

AChR clustering

activity

of agrin transfected

cells To assay the ability of different forms of agrin to cluster AChRs, cDNAs were ligated together into a linear sequence predicted to encode a full length agrin protein. The constructs were transfected into either CHO or COS cells [27*]. Antibodies generated to either aminoterminal, middle, or carboxy-terminal regions of the predicted protein recognized a molecule of approximately 205kD in western blotting experiments on transfected cells. Staining with the antibodies in the presence of saponin revealed intracellular immunoreactivity in a retic-

The organization of the neuromuscular junction Rupp, Hoch, Campanelli,

ular pattern characteristic of the endoplasmic reticulum and the Golgi apparatus. In the absence of saponin, the antibodies stained the surface of the transfected cells, particularly at sites of cell-cell contact. Electron microscope immunohistochemical studies also reveal surface labeling of transfected cells. The gold particles are often associated with electron dense material on the CHO cell surfaces suggestive of extracellular matrix. It is thus clear that the agrin sequence delined by the analysis of the rat cDNAs encodes a secreted protein, and it is likely that the molecule assembles into a matrix on the surface of tmnsfected cells [ 27.1. When transfected cells are co-cultured with muscle fibers, clusters of AChRs are often localized to sites of contact between the muscle fiber and the agrir-expressing cells. In contrast with the original assay used to purify agrin, in this system no increase in the number of AChR clusters is seen outside the sites of cell-cell contact. Several possibilities may account for this. First, the original molecules purified from marine rays were probably proteolytic fragments of a longer molecule representing the major in vivo form. These longer forms of the protein may not be active when applied in a bathing medium, because the context of presentation is not appropriate. Second, most of the protein in transfected cells is attached to the plasma membrane or extracellular matrix, raising the possibility that the co-cultures do not generate high enough concentrations of the molecules to be functional outside of ce&cell contact sites. In both CHO and COS cells the form of the protein recovered from the media was found to be 1520 kD smaller than the cell-attached form. Thus, proteolysis may inactivate the molecules in co-culture media. Third, experiments suggest that the rat muscle cultures dilfer in some ways from chick muscle cultures. AChR clusters tend to be larger in the rat, suggesting that species differences may be important in understanding the mechanisms of agrin-induced AChR clustering. Fourth, it is possible that other forms of agrin are more active in the soluble clustering assay. These forms could be generated by alternative splicing or represent the products of different genes. Although alternatively spliced forms appear to act differentially in chick, preliminary data in the rat have not revealed any differences in the activity among the various known spliced forms of the protein when tested on rat myotubes in culture. The clustering activity observed in co-cultures with transfected cells is most likely to mimic the in vivo situation, and this system will provide insights useful in resolving the roles that different forms of agrin play in synapse development.

The mechanisms

of AChR clustering

How does agrin act to cluster AChRs at synaptic sites? It is not possible to answer this question deiinitively; however, several recent reports are relevant to this issue. It has been known for some time that assembled AChRs co-purify with a protein associated with the intracellular side of the receptor [ 281. The precise function of this molecule, known as the ‘43 kD protein’, is unknown; one D

Kreiner

and

Scheller

hypothesis is that it acts to organize AChRs through linkages with the cytoskeleton [29]. Two studies have explored the effects of the 43 kD protein on AChR clusters using Xenopus oocyte and fibroblast expression systems [30-,31*]. Both groups report that co-expression of the 43 kD protein with the four AChR subunits increases the number of receptor clusters, demonstrating at least a facilitatory role for this protein in receptor clustering. The AChR is phosphorylated, and it is attractive to speculate that this modification plays a role in receptor clustering. In support of this hypothesis, addition of agrin to chicken myotubes in culture results in phosphotylation of ‘Iyr residues on the P-subunit of the AChR [32*]. This phosphorylation precedes cluster formation; the time course is consistent with a causal relationship, and inhibitors of tyrosine kinases block’the clustering

[13*1.

,-

’;.

Paradoxically, phosphotyrosine cannot ‘be. detected with antibodies in AChR aggregates in “in vivo mammalian systerns until 2 weeks after cluster formation [33*]. One explanation is that the technique is not adequate to detect a level of phosphotyrosine sufficient to induce AChR clusters. An alternative explanation is that phosphotylation is not relevant to clustering; instead, the tyrosine phosphotylation may be important in stabilizing AChR clusters, or in directing aspects of endocytosis. While the experiments are not definitive at this stage, it is likely that both phosphorylation and the 43kD protein have roles in localizing AChRs to synaptic sites. Perhaps agrin acts through a tyrosine kinase receptor on the muscle cell surface to regulate association of the 43 kD protein with AChRs. Experiments are in progress to test these hypotheses. While the proposals above may account for the formation of clusters, it is necessary to further explain the formation of high density receptor aggregates at the sites of contact between nerve and muscle. Addition of agrin to chicken muscle fiber cultures induces the formation of what appear to be randomly dispersed clusters. In cells transfected with a rat agrin gene, aggregates appear at sites of contact similar to those seen in nerve induced clustering. As agrin-induced clusters on chick myotubes in culture are rich in acetylcholinesterase, 43 kD protein, heparan sulfate proteoglycan, and agrin itself, it is unlikely that direct interactions of agrin with all of these molecules can account for the observed phenomena. A local signaling system could play a major role in the mechanism by which agrin induces clusters of receptors and other molecules. If this is correct, it is unlikely that a diffusible second messenger is produced. Perhaps as AChRs migrate in the plasma membrane, they enter the radius of influence of an agrin-activated receptor system, and are subsequently lixed at or near those positions by anchoring to the cytoskeleton via the 43 kD protein.

Conclusion Many of the unresolvd issues in synapse development, including those discussed above, are now approachable

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using current molecular, genetic, and biochemical techniques. While agrin is likely to be a key player in synapse development, this complex process is almost certainly regulated by the reciprocal exchange of pre- and postsynaptic information. An understanding of how this information is modulated by neuronal activity may begin to suggest molecular mechanisms that could account for the modulation of synaptic efficacy thought to be important in the storage of memories.

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the Accumulation of Acetylchollne Receptor at Developing Chick Neuromuscular Junction. G&i Spring Harb Symp Quunt Biol 1990, LV:397-@6. Thus review otters a comprehensive update of the biochemical and molecular data on ARIA The identification and puriIication of ARIA from total chick brain extracts is described. Protein sequence data have been successfully used for the cloning of a candidate ARIA encoding cDNA. This cDNA clone shows homology to the mammalian priori protein gene.

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FROEHNER SC, LUETJE CW, Scow PB, PATRICK J: The Postsynaptic 43 K Protein Clusters Muscle Nicotinic Acetylcholine Receptors in Xenopus Oocytes. Neuron 1990, 5:4OwlO. In this study, Xenqus oocytes were co-injected with RNAs specific for alI nicotinic AChRs. Oocytes injected exclusively with AChR subunit RNAs displayed evenly distributed receptors on the surface. Interestingly, injection of the 43 kD polypcptide RNA alone caused aggregates of this protein. The results suggest that the 43 kD protein plays an tiportant role in the clustering of surface nicotinic AChRs. 30. .

PHILLIPS WD, KOPTAC, BUXJNT P, GARDNER PD, STEINBACH JH, MERUEJP: ACh Receptor-Rich Domains Organized in Fibroblasts by Recombinant 43Xilodalton Protein. Science 1991, 251:56&570. Fibroblasts stably transfected with all the AChR subunits show receptor immunoreactivity dispersed on the cell surface. Transient transfection of these cells with an expression vector encoding the 43kD protein causes the AChRs to aggregate into large clusters. These data indicate that the 43 kD protein can induce AChR clustering by interacting with the receptor. 31. .

32. .

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Kreiner

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Addition of agrin to cultured chick myotubes induces AChR phospho. ryiation of the fi-, y-, and S-receptor subunits. The agrin-induced AC~R aggregation and p-subunit phosphotylation cannot be blocked with a protein serine kinase inhibitor. Prevention of agrin-induced aggregation by low pH, phorbol 1,2-myristate 1,3-acetate (TPA), or polyanions also prevents p-subunit phosphotyiation. These results indicate that tyrosine phospholyiation of the receptor p-subunit is modulated by AChR aggregation. Qu 2, MORTIZ E, HUGANIR RL: Regulation of mosine Phosphorylation of the Nicotinic Acetylcholine Receptor at the Rat Neuromuscular Junction. Neuroscience 1990, 4:367-378. AChR phospholyiation levels on Tyr residues are studied with immunocytochemical and immunoblotting techniques. Tyr phosphorylation at the rat NMJ can be shown between postnatal days 7-14, and is colocalized with AChR immunoreacritity. The level of v phosphorylation is reduced in denervated muscles, suggesting thit innervation plays a role in receptor Tyr phosphotylation during development and regeneration. 33. .

F Rupp, W Hoch, JT CampaneUi, T Kreiner ani RFI Scheller, Howard Hughes Medical Institute, Depanment ofkoiecular and Cellular Physiology, Beckman Center, Stanford University, Stanford, California 94305, USA

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