Structural insights into the assembly of the agrin/LRP4/MuSK signaling complex.

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability.

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

Antibodies to Full-Length Agrin Protein in Chinese Patients With Myasthenia Gravis.

This study aimed to establish a cell-based assay (CBA) for the detection of agrin antibodies (Agrin-Ab) to explore the clinical features of agrin antibody-positive Chinese patients with myasthenia gravis (Agrin-MG). We developed a CBA based on the human full-length agrin protein expressed in HEK293T cells for the reliable and efficient detection of Agrin-Ab. Clinical data and serum samples were collected from 1948 MG patients in 26 provinces in China. The demographic and clinical features of Agrin-MG patients were compared with those of other MG patient subsets. Eighteen Agrin-MG cases were identified from 1948 MG patients. Nine patients were Agrin-Ab positive, and nine were AChR-Ab and Agrin-Ab double-positive (Agrin/AChR-MG). Eleven (61.11%) patients were males older than 40 years of age. The initial symptom in 13 (81.25%) cases was ocular weakness. Occasionally, the initial symptom was limb-girdle weakness (two cases) or bulbar muscle weakness (one case). Agrin-MG patients demonstrated slight improvement following treatment with either acetylcholinesterase inhibitor or prednisone; however, the combination of the two drugs could effectively relieve MG symptoms. In China, Agrin-MG demonstrated seropositivity rates of 0.92%. These patients were commonly middle-aged or elderly men. The patients usually presented weakness in the ocular, bulbar, and limb muscles, which may be combined with thymoma. These patients have more severe diseases, although the combination of pyridostigmine and prednisone was usually effective in relieving symptoms.

Decreased Serum Levels of C-Terminal Agrin in Postmenopausal Women Following Resistance Training.

Elevated circulating C-terminal agrin fragment (CAF) is a marker of neuromuscular junction degradation and sarcopenia. This study sought to determine if resistance training (RT) impacted the serum levels of CAF in perimenopausal (PERI-M) and postmenopausal (POST-M) women. A total of 35 women, either PERI-M or POST-M, participated in 10 weeks of RT. Body composition, muscle strength, and serum estradiol and CAF were determined before and after the RT. The data were analyzed with two-way analysis of variance (p ≤ .05). Upper body and lower body strength was significantly increased, by 81% and 73% and 86% and 79% for the PERI-M and POST-M participants, respectively; however, there were no significant changes in body composition. Estradiol was significantly less for the POST-M participants at pretraining compared with the PERI-M participants. CAF moderately increased by 22% for the PERI-M participants in response to RT, whereas it significantly decreased by 49% for the POST-M participants. Ten weeks of RT reduced the circulating CAF in the POST-M women and might play a role in attenuating degenerative neuromuscular junction changes.

Pathogenic effects of agrin V1727F mutation are isoform specific and decrease its expression and affinity for HSPGs and LRP4.

Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y-z- agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y-z- agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y-z- agrin function in other tissues appears preserved.

An affinity-based approach to engineer laminin-presenting cell instructive microenvironments.

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices.

Interaction studies of a protein and carbohydrate system using an integrated approach: a case study of the miniagrin-heparin system.

The major challenges in biophysical characterization of human protein-carbohydrate interactions are obtaining monodispersed preparations of human proteins that are often post-translationally modified and lack of detection of carbohydrates by traditional detection systems. Light scattering (dynamic and static) techniques offer detection of biomolecules and their complexes based on their size and shape, and do not rely on chromophore groups (such as aromatic amino acid sidechains). In this study, we utilized dynamic light scattering, analytical ultracentrifugation and small-angle X-ray scattering techniques to investigate the solution properties of a complex resulting from the interaction between a 15 kDa heparin preparation and miniagrin, a miniaturized version of agrin. Results from dynamic light scattering, sedimentation equilibrium, and sedimentation velocity experiments signify the formation of a monodisperse complex with 1:1 stoichiometry, and low-resolution structures derived from the small-angle X-ray scattering measurements implicate an extended conformation for a side-by-side miniagrin‒heparin complex.

Engineered agrin attenuates the severity of experimental autoimmune myasthenia gravis.

INTRODUCTION: Agrin is essential for the formation and maintenance of neuromuscular junctions (NMJs). NT-1654 is a C-terminal fragment of mouse neural agrin. In this study, we determined the effects of NT-1654 on the severity of experimental autoimmune myasthenia gravis (EAMG). METHODS: EAMG was induced in female Lewis rats by immunization with the Torpedo acetylcholine receptor (tAChR) and complete Freund's adjuvant (CFA). NT-1654 was dissolved in phosphate-buffered saline (PBS) and injected daily subcutaneously into tAChR immunized rats during the first 10 days after immunization, and then every other day for the following 20 days. RESULTS: We showed that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. DISCUSSION: We demonstrated that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. Muscle Nerve 57: 814-820, 2018.

The Basement Membrane Proteoglycans Perlecan and Agrin: Something Old, Something New.

Several members of the proteoglycan family are integral components of basement membranes; other proteoglycan family members interact with or bind to molecular residents of the basement membrane. Proteoglycans are polyfunctional molecules, for they derive their inherent bioactivity from the amino acid motifs embedded in the core protein structure as well as the glycosaminoglycan (GAG) chains that are covalently attached to the core protein. The presence of the covalently attached GAG chains significantly expands the "partnering" potential of proteoglycans, permitting them to interact with a broad spectrum of targets, including growth factors, cytokines, chemokines, and morphogens. Thus proteoglycans in the basement membrane are poised to exert diverse effects on the cells intimately associated with basement membranes.

C-terminal agrin fragment is inversely related to neuromuscular fatigue in older men.

INTRODUCTION: The aim of this study was to examine the relationship between serum C-terminal agrin fragment (CAF) concentrations and neuromuscular fatigue in older adults. METHODS: Twenty-two healthy older men and women volunteered for this study. Resting fasted blood samples were collected and prepared for measurement of serum CAF concentration by a commercially available ELISA kit. The onset of neuromuscular fatigue was measured by monitoring electromyographic fatigue curves from the vastus lateralis muscle using the physical working capacity at fatigue threshold (PWCFT ) test. RESULTS: A significant inverse correlation for men was observed between CAF and PWCFT (r = -0.602; P = 0.05), but not for women (r = 0.208; P = 0.54). After controlling for age and body mass index, significant correlations (r = -0.69; P = 0.042) remained for men, but not for women (r = 0.12; P = 0.76). CONCLUSIONS: These data suggest that serum CAF concentrations were significantly related to the onset of neuromuscular fatigue independent of age and BMI in men only.

C-terminal agrin fragment--a new fast biomarker for kidney function in renal transplant recipients.

BACKGROUND: The C-terminal agrin fragment (CAF) is a cleavage product of agrin, the major proteoglycan of the glomerular basement membrane. This article studies if CAF could serve as a biomarker for renal function in renal transplant recipients. MATERIAL AND METHODS: We measured serum CAF and creatinine concentrations and calculated estimated glomerular filtration rate (eGFR) (MDRD) in 96 healthy individuals and in 110 end-stage renal disease patients undergoing kidney transplantation before and after transplantation. Correlation between CAF and creatinine concentrations/eGFR was calculated as within-patient (cWP) and between-patient correlations (cBP). Moreover, we evaluated the association of CAF with delayed graft function (DGF). The diagnostic value of CAF for early detection of DGF compared to creatinine was evaluated by receiver operating characteristics (ROC) analysis. RESULTS: CAF concentrations strongly correlated with creatinine (r = 0.86 (cWP), r = 0.74 (cBP)) and eGFR (MDRD) (r = 0.86 (cWP), r = 0.77 (cBP)). Pre-transplant (pre-Tx) CAF concentrations were 19-fold higher than in healthy individuals (1,115.0 (258.4-3,990.0) vs. 56.6 (20.0-109.5) pM). After transplantation, CAF decreased significantly faster than creatinine (postoperative days 1-3 (POD 1-3): 562.8 (101.6-2,113.0) pM; creatinine: pre-Tx 6.9 (3.1-15.7), POD 1-3: 6.4 (1.7-12.7) mg/dl, p < 0.001). Stable concentrations were reached 1-3 months after transplantation for CAF and creatinine (CAF 145.1 (6.7-851.0) pM; creatinine 1.6 (0.7-8.0) mg/dl). CAF concentrations at POD 1-3 were significantly associated with DGF and outperformed creatinine in early detection of DGF (area under the curve (AUC) CAF 80.7% (95% CI 72.3-89.1%) vs. AUC creatinine 71.3% (95% CI 61.8-81.1%), p = 0.061). CONCLUSION: CAF is a promising new and fast biomarker for kidney function and may serve as a new tool for the early detection of DGF.

Structure and activation of MuSK, a receptor tyrosine kinase central to neuromuscular junction formation.

MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.

Nephronectin binds to heparan sulfate proteoglycans via its MAM domain.

Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase µ (MAM) domain. Nephronectin has been shown to interact with α8ß1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.

Structural mechanisms of the agrin-LRP4-MuSK signaling pathway in neuromuscular junction differentiation.

The neuromuscular junction (NMJ) is the most extensively studied model of neuronal synaptogenesis. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in the differentiation of NMJs. AChR clustering and postsynaptic differentiation is orchestrated by sophisticated interactions among three proteins: the neuron-secreted proteoglycan agrin, the co-receptor LRP4, and the muscle-specific receptor tyrosine kinase MuSK. LRP4 and MuSK act as scaffolds for multiple binding partners, resulting in a complex and dynamic network of interacting proteins that is required for AChR clustering. In this review, we discuss the structural basis for NMJ postsynaptic differentiation mediated by the agrin-LRP4-MuSK signaling pathway.

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane.

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3ß, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

The disturbed blood-brain barrier in human glioblastoma.

The aim of this article is to describe alterations of the blood-brain barrier (BBB) in gliomas. The main clinical problem of human gliomas is the edematous swelling and the dramatic increase of intracerebral pressure, also compromising healthy areas of the brain. According to our concept, one of the main reasons on the cellular level for these clinical problems is the loss or reduction of astroglial polarity. Astroglial polarity means the specific accumulation of potassium and water channels in the superficial and perivascular astroglial endfeet membranes. The most important water channel in the CNS is the astroglial water channel protein aquaporin-4 (AQP4) which is arranged in a morphologically spectacular way, the so-called orthogonal arrays of particles (OAPs) to be observed in freeze-fracture replicas. In brain tumors, but also under conditions of trauma or inflammation, these OAPs are redistributed to membrane domains apart from endfeet areas. Probably, this dislocation might be due to the degradation of the proteoglycan agrin by the matrix metalloproteinase 3 (MMP3). Agrin binds to the dystrophin-dystroglycan-complex (DDC), which in turn is connected to AQP4. As a consequence, agrin loss may lead to a redistribution of AQP4 and a compromised directionality of water transport out of the cell, finally to cytotoxic edema. This in turn is hypothesized to lead to a breakdown of the BBB characterized by disturbed tight junctions, and thus to the development of vasogenic edema. However, the mechanism how the loss of polarity is related to the disturbance of microvascular tight junctions is completely unknown so far.

Structural basis of agrin-LRP4-MuSK signaling.

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.

LG2 agrin mutation causing severe congenital myasthenic syndrome mimics functional characteristics of non-neural (z-) agrin.

We describe a severe form of congenital myasthenic syndrome (CMS) caused by two heteroallelic mutations: a nonsense and a missense mutation in the gene encoding agrin (AGRN). The identified mutations, Q353X and V1727F, are located at the N-terminal and at the second laminin G-like (LG2) domain of agrin, respectively. A motor-point muscle biopsy demonstrated severe disruption of the architecture of the neuromuscular junction (NMJ), including: dispersion and fragmentation of endplate areas with normal expression of acetylcholinesterase; simplification of postsynaptic membranes; pronounced reduction of the axon terminal size; widening of the primary synaptic cleft; and, collection of membranous debris material in the primary synaptic cleft and in the subsynaptic cytoplasm. Expression studies in heterologous cells revealed that the Q353X mutation abolished expression of full-length agrin. Moreover, the V1727F mutation decreased agrin-induced clustering of the acetylcholine receptor (AChR) in cultured C2 muscle cells by >100-fold, and phosphorylation of the MuSK receptor and AChR beta subunit by ~tenfold. Surprisingly, the V1727F mutant also displayed increased binding to α-dystroglycan but decreased binding to a neural (z+) agrin-specific antibody. Our findings demonstrate that agrin mutations can associate with a severe form of CMS and cause profound distortion of the architecture and function of the NMJ. The impaired ability of V1727F agrin to activate MuSK and cluster AChRs, together with its increased affinity to α-dystroglycan, mimics non-neural (z-) agrin and are important determinants of the pathogenesis of the disease.

A valid mouse model of AGRIN-associated congenital myasthenic syndrome.

Congenital myasthenic syndromes (CMS) are inherited diseases affecting the neuromuscular junction (NMJ). Mutations in AGRIN (AGRN) and other genes in the AGRIN signaling pathway cause CMS, and gene targeting studies in mice confirm the importance of this pathway for NMJ formation. However, these mouse mutations are complete loss-of-function alleles that result in an embryonic failure of NMJ formation, and homozygous mice do not survive postpartum. Therefore, mouse models of AGRIN-related CMS that would allow preclinical testing or studies of postnatal disease progression are lacking. Using chemical mutagenesis in mice, we identified a point mutation in Agrn that results in a partial loss-of-function allele, creating a valid model of CMS. The mutation changes phenylalanine 1061 to serine in the SEA domain of AGRIN, a poorly characterized motif shared by other extracellular proteoglycans. NMJs in homozygous mice progressively degrade postnataly. Severity differs with genetic background, in different muscles, and in different regions within a muscle in a pattern matching mouse models of motor neuron disease. Mutant NMJs have decreased acetylcholine receptor density and an increased subsynaptic reticulum, evident by electron microscopy. Synapses eventually denervate and the muscles atrophy. Molecularly, several factors contribute to the partial loss of AGRIN's function. The mutant protein is found at NMJs, but is processed differently than wild-type, with decreased glycosylation, changes in sensitivity to the protease neurotrypsin and other proteolysis, and less efficient externalization and secretion. Therefore, the Agrn point mutation is a model for CMS caused by Agrn mutations and potentially other related neuromuscular diseases.

Evidence for self-association of a miniaturized version of agrin from hydrodynamic and small-angle X-ray scattering measurements.

Hydrodynamic studies of miniagrin indicate a molar mass that is 20% larger than the value calculated from the sequence of this genetically engineered protein. Consistent with this finding is the negative sign and also the magnitude of the second virial coefficient obtained from small-angle X-ray scattering measurements. The inference that miniagrin reversibly self-associates is confirmed by a sedimentation equilibrium study that yields an equilibrium constant of 0.24 L/g for a putative monomer-dimer interaction. Finally, Guinier analysis of the small-angle X-ray scattering (SAXS) results yields concentration-dependent values for the radius of gyration that may be described by the monomer-dimer model and respective R(g) values of 40 and 105 Å for the monomeric and dimeric miniagrin species. Although intermolecular protein interactions are endemic in the events leading to acetylcholine receptor aggregation by agrin, the matrix proteoglycan of which miniagrin is a miniaturized model, this investigation raises the possibility that agrin may itself self-associate.