Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors.

At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, beta-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyn-deficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and beta-dystroglycan, and for the agrin-induced increase in association with MuSK, but not for constitutive interactions with MuSK and src-related kinases. In rapsyn -/- myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, whereas phosphorylation of the AChR beta subunit, both constitutive and agrin-induced, was strongly reduced. These results show first that aneural myotubes contain preassembled AChR protein complexes that may function in the assembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utrophin glycoprotein complex.

Altered glycosylation sites of the delta subunit of the acetylcholine receptor (AChR) reduce alpha delta association and receptor assembly.

We have used mutagenesis to investigate the potential N-glycosylation sites in the delta subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn76, Asn143, and Asn169, only the first two were glycosylated when the delta subunit was expressed in COS cells. Because the heterologously expressed delta subunit was similar in its properties to that expressed in C2 muscle cells, the sites of glycosylation are likely to be the same in both cases. In COS cells, mutations of the delta subunit that prevented glycosylation at either of the sites did not change its metabolic stability nor its steady-state level. These results are in contrast to those found previously for the alpha subunit, in which glycosylation at a single site metabolically stabilized the polypeptide (Blount, P., and Merlie, J. P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the delta subunit that prevented glycosylation, however, decreased its ability to form an alpha delta heterodimer when the alpha and delta subunit were expressed together. When all four subunits of the AChR (alpha, beta, delta, and epsilon) were coexpressed, mutation of the delta subunit to prevent glycosylation resulted in a reduced amount of fully assembled AChR and reduced surface AChR levels, consistent with the role of the heterodimer in the assembly reaction. These results suggest that glycosylation of the delta subunit at both Asn76 and Asn143 is needed for its efficient folding and/or its subsequent interaction with the alpha subunit.

Metabolic stabilization of muscle nicotinic acetylcholine receptor by rapsyn.

Although the metabolic half-life of muscle endplate acetylcholine receptor (AChR) changes during development and after denervation in the adult, little is known about the molecular mechanisms that influence receptor stability. We have investigated the effect on AChR turnover of its interaction with rapsyn, a 43 kDa peripheral membrane protein that is closely associated with the AChR in muscle cells and is required for its clustering at endplates. Both in transfected COS cells and in cultured myotubes from rapsyn-negative and rapsyn-positive mice, we have found that the presence of rapsyn slows the turnover of AChRs by as much as twofold. The effect was similar for both embryonic (alpha2betadeltagamma) and adult (alpha2betadeltaepsilon) AChRs and for AChRs whose beta subunit lacked a putative tyrosine phosphorylation site. Neither colchicine nor cytochalasin D altered AChR turnover or prevented the rapsyn effect. Mutant rapsyn proteins whose N-terminal myristoylation signal was eliminated, or whose C terminus or zinc-finger domains were deleted, failed to change the rate of receptor turnover. Each of these mutations affects the association of the AChR with rapsyn, suggesting that AChR stability is altered by interaction between the two proteins. Our results suggest that, in addition to its role in AChR clustering, rapsyn also functions to metabolically stabilize the AChR.

Laminin-induced acetylcholine receptor clustering: an alternative pathway.

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

Functional interaction of Src family kinases with the acetylcholine receptor in C2 myotubes.

Tyrosine phosphorylation of the beta subunit of the acetylcholine receptor (AChR) has been postulated to play a role in AChR clustering during development of the neuromuscular junction. We have investigated the mechanism of this phosphorylation in mammalian C2 myotubes and report that the tyrosine kinase Src binds and phosphorylates glutathione S-transferase fusion proteins containing the N-terminal half of the cytoplasmic loop of the beta subunit. No binding occurs to the related kinases Fyn or Yes or to the corresponding regions from the gamma and delta subunits. Furthermore, AChRs affinity-isolated from C2 myotubes using alpha-bungarotoxin-Sepharose were specifically associated with Src and Fyn and had tyrosine-phosphorylated beta subunits. We suggest that AChRs are initially phosphorylated by Src and subsequently bind Fyn in a phosphotyrosine-dependent manner. These interactions are likely to play an important role in construction of the specialized postsynaptic membrane during synaptogenesis.

Assembly of the nicotinic acetylcholine receptor. The first transmembrane domains of truncated alpha and delta subunits are required for heterodimer formation in vivo.

To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal fragments of the alpha and delta subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and to form a ligand-binding site. Truncated fragments of the alpha subunit that include all, part, or none of the first transmembrane domain (M1) folded to acquire alpha-bungarotoxin binding activity. Neither the full-length alpha subunit nor any of the fragments were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into the medium. When coexpressed with the delta subunit, the alpha subunit fragment possessing M1 formed a heterodimer containing a ligand-binding site, but shorter fragments, which lack transmembrane segments, did not associate with the delta subunit. N-terminal delta subunit fragments gave similar results. An N-terminal delta subunit fragment that contains M1 associated with the alpha subunit to form a heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association of truncated N-terminal alpha and delta subunits into a heterodimer with high affinity ligand binding activity.

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site.

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.

Formation of a ligand-binding site for the acetylcholine receptor in vitro.

Investigation of the mechanisms by which the subunits of ligand-gated ion channels fold and associate to form oligomers has been hampered by the lack of an in vitro system in which these reactions occur. We have established conditions in a rabbit reticulocyte translation system supplemented with canine pancreatic microsomes under which the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) fold and assemble to form a heterodimer with a cholinergic binding site comparable with that found in the intact AChR. Folding of the alpha subunit was followed by its ability to bind alpha-bungarotoxin. Folding efficiency was highly sensitive to changes in the redox potential of the translation medium and was favored by an oxidizing environment. Acquisition of the toxin binding conformation required N-linked core glycosylation but not oligosaccharide trimming, suggesting that oligosaccharide-dependent interaction of chaperones with the alpha subunit is not essential for correct subunit folding. The conformationally mature alpha subunit specifically associated with the delta subunit but not the beta subunit to form a heterodimer with a high affinity ligand-binding site. These data demonstrate, for the first time, correct folding and assembly of the AChR subunits in an in vitro system.

Altered glycosaminoglycan chain structure in a variant of the C2 mouse muscle cell line.

Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of 35SO4 into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation.

Neural agrin activates a high-affinity receptor in C2 muscle cells that is unresponsive to muscle agrin.

During synaptogenesis, agrin, released by motor nerves, causes the clustering of acetylcholine receptors (AChRs) in the skeletal muscle membrane. Although muscle alpha-dystroglycan has been postulated to be the receptor for the activity of agrin, previous experiments have revealed a discrepancy between the biological activity of soluble fragments of two isoforms of agrin produced by nerves and muscles, respectively, and their ability to bind alpha-dystroglycan. We have determined the specificity of the signaling receptor by investigating whether muscle agrin can block the activity of neural agrin on intact C2 myotubes. We find that a large excess of muscle agrin failed to inhibit either the number of AChR clusters or the phosphorylation of the AChR induced by picomolar concentrations of neural agrin. These results indicate that neural, but not muscle, agrin interacts with the signaling receptor. Muscle agrin did block the binding of neural agrin to isolated alpha-dystroglycan, however, suggesting either that alpha-dystroglycan is not the signaling receptor or that its properties in the membrane are altered. Direct assay of the binding of muscle or neural agrin to intact myotubes revealed only low-affinity binding. We conclude that the signaling receptor for agrin is a high-affinity receptor that is highly specific for the neural form.

Building synapses: agrin and dystroglycan stick together.

A major effort of the past decade for those studying synaptic development has been to identify the molecular signals whose carefully choreographed exchange between pre- and postsynaptic cells regulates the local differentiation of each cell to form the mature synapse. Now that several of these factors [agrin, ACh-receptor inducing activity (ARIA) and calcitonin gene-related peptide] have been identified and isolated, efforts have moved toward understanding their receptors and the intracellular signaling pathways by which the factors achieve their effects. One of the most intensively studied of the synaptic signaling molecules is agrin, a large protein synthesized and released by motor neurons that induces ACh receptors and other synaptic molecules in muscle cells to accumulate at the sites of nerve contact. Recent efforts to discover the agrin receptor have led to a surprising conclusion: the only agrin-binding component so far detected in muscle cells is dystroglycan, an extracellular protein that is part of the complex of proteins associated with dystrophin, and its homologue, utrophin. Because dystroglycan binds laminin, and dystrophin binds actin, the complex containing these two proteins is thought to link the extracellular matrix to the cytoskeleton. Those interested in synapses are now pondering whether dystroglycan has a new and unexpected role as a signaling receptor for agrin-induced ACh-receptor clustering, whether it serves as an auxiliary for another receptor, or whether it serves as a receptor for an entirely different agrin-mediated function.

Amino- and carboxyl-terminal domains specify the identity of the delta subunit in assembly of the mouse muscle nicotinic acetylcholine receptor.

We have used transient expression in COS cells of the subunits of the nicotinic acetylcholine receptor (AChR) from mouse skeletal muscle to investigate the role of transmembrane and cytoplasmic domains of the delta subunit in assembly of the AChR. When chimeric subunits whose extracellular amino- and carboxyl-terminal domains were from the delta subunit and whose transmembrane and cytoplasmic domains were from either the beta, gamma, or epsilon subunit were expressed with alpha, beta, and epsilon subunits, alpha-bungarotoxin-binding activity appeared on the surface of the transfected cells. The resulting receptor complexes each had sedimentation constants resembling those of the native AChR, consistent with a pentameric structure. Further investigation of the delta beta chimeric subunit showed that it formed a heterodimer with the alpha subunit and that the resulting subunit bound d-tubocurarine with an affinity similar to that of the alpha delta heterodimer; delta beta also formed a heterodimer with a form of the alpha subunit that is truncated after the first transmembrane domain. A heterodimer formed from the epsilon beta and alpha subunits also bound d-tubocurarine with an affinity similar to that of the alpha epsilon heterodimer. When both epsilon beta and delta beta subunits were substituted for the epsilon and delta subunits, respectively, a receptor complex was formed whose structure appeared to be alpha 2 beta(epsilon beta)(delta beta). These results show that, as with the epsilon subunit, the identity of the delta subunit in AChR assembly arises from the extracytoplasmic domains of the subunit.

Dystroglycan binds nerve and muscle agrin.

Neurally released agrin is thought to cluster acetylcholine receptors (AChRs) and other synaptic proteins in the postsynaptic membrane during synaptogenesis at the neuromuscular junction. We have examined the binding of nerve and muscle agrins, which have dramatically different abilities to cluster AChRs, to the membrane proteins of Torpedo electric organ and C2 myotubes. Both bound with approximately nanomolar affinity to a single component identified as alpha-dystroglycan: agrin binding was blocked by antibodies to alpha-dystroglycan, and agrin bound to purified alpha-dystroglycan. Dystroglycan was altered in two genetic variants of C2 muscle cells that fail to form spontaneous clusters of AChRs and that show a diminished response to agrin. Antibodies that blocked alpha-dystroglycan binding, however, failed to block the clustering of AChRs by neural agrin. Although alpha-dystroglycan is the major agrin-binding protein in Torpedo and myotube membranes, its physiological role is unclear.