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PubMed1995

An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix.

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.

Agrin↗
PubMed2000

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

Agrin↗
PubMed2001

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

Agrin↗
PubMed2003

The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons.

Agrin is a motor neuron-derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron-neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron-neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.

Agrin↗
PubMed2000

Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture.

Agrin secreted by motor neurons is a critical signal for postsynaptic differentiation at the developing neuromuscular junction. We used cultures of chick ventral spinal cord neurons with rat myotubes and immunofluorescence with species-specific antibodies to determine the distribution of agrin secreted by neurons and compare it to the distribution of agrin secreted by myotubes. In addition, we determined the distribution of agrin secreted by isolated chick ventral spinal cord neurons and rat motor neurons grown on a substrate that binds agrin. In cocultures, neuronal agrin was concentrated along axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in synaptogenesis. Smaller amounts of agrin were found on dendrites and cell bodies and rarely were associated with AChR aggregation. Muscle agrin, recognized by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neuronal agrin. In cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the first 2-3 days in culture. In older cultures, agrin immunoreactivity was markedly more intense around axons than dendrites, indicating that motor neurons possess an intrinsic, developmentally regulated program to target agrin secretion to axons.

Agrin↗
PubMed1999

Identification of extracellular matrix ligands for the heparan sulfate proteoglycan agrin.

Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development.

Agrin↗
PubMed1999

Expression, distribution and ultrastructural localization of the synapse-organizing molecule agrin in the mature avian retina.

At the vertebrate neuromuscular junction the extracellular matrix molecule agrin is responsible for the formation, maintenance and regeneration of most if not all postsynaptic specializations. Several agrin isoforms are generated by alternative splicing which differ in their function and which are all expressed in the CNS. To analyse the role of agrin in the CNS, we investigated the expression and ultrastructural localization of agrin in the posthatched chick retina. In situ hybridization revealed the presence of agrin mRNA in all cellular layers of the mature retina, indicating that most if not all major retinal cell types synthesize agrin. Pan-specific as well as isoform-specific antiagrin antisera stained the optic fibre layer and the outer plexiform layer. However, only the pan-specific antiserum additionally stained the inner limiting membrane. Immunoelectron microscopy showed that in the optic fibre layer agrin was associated with ganglion cell axons and that at least part of this agrin corresponds to a neuronal isoform of agrin. In the outer plexiform layer, agrin was localized in the cleft between the photoreceptor terminals and the invaginating horizontal and bipolar cell dendrites. In the synapse-containing inner plexiform layer both antisera revealed punctate immunoreactivity. This staining corresponded to agrin concentrated in the synaptic cleft of conventional synapses as determined by preembedding immunoelectron microscopy. Agrin is thus concentrated at mature interneuronal synapses as it is at the neuromuscular junction, consistent with a role of agrin during formation and/or maintenance of synapses in the CNS.

Agrin↗
PubMed1995

Identification of a novel alternatively spliced agrin mRNA that is preferentially expressed in non-neuronal cells.

A novel agrin isoform was identified based on the isolation of an agrin cDNA from E9 chick brain that lacked 21 base pairs (bp) in the NH2-terminal encoding region of the agrin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) of E9 chick brain mRNA confirmed the existence of this agrin isoform in brain, although the novel splice variant represents a minor fraction of agrin mRNA in brain. However, upon analysis of chick brain astrocyte mRNA, smooth muscle mRNA, and cardiac muscle mRNA by RT-PCR, we show that this novel agrin isoform is the predominant agrin isoform in these non-neuronal cell populations. We extended our analyses to examine the expression of this agrin mRNA isoform during chick development and show that the agrin mRNA lacking this 21-bp exon is up-regulated with brain development, consistent with the increase in glial number during brain development, while the agrin isoform that does not undergo splicing and thus contains the 21-bp exon is down-regulated in brain development. Because the 21-bp exon is inserted in the region of chick agrin which encodes the putative signal sequence of agrin, with the signal peptidase site immediately preceding the putative first amino acid of the mature protein being deleted as a result of splicing, these data raise the interesting possibility that the presence or absence of this alternatively spliced exon may differentially regulate processing of the agrin protein in neuronal and non-neuronal cells, respectively.

Agrin↗
PubMed1998

Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Agrin↗
PubMed1988

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester.

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.

Agrin↗
PubMed1996

Non-neural agrin codistributes with acetylcholine receptors during early differentiation of Torpedo electrocytes.

Agrin, an extracellular matrix protein synthesized by nerves and muscles is known to promote the clustering of acetylcholine receptors and other synaptic proteins in cultured myotubes. This observation suggests that agrin may provide at least part of the signal for synaptic specialization in vivo. The extracellular matrix components agrin, laminin and merosin bind to alpha-dystroglycan, a heavily glycosylated peripheral protein part of the dystrophin-glycoprotein complex, previously characterized in the sarcolemma of skeletal and cardiac muscles and at the neuromuscular junction. In order to understand further the function of agrin and alpha DG in the genesis of the acetylcholine receptor-rich membrane domain, the settling of components of the dystrophin-glycoprotein complex and agrin was followed by immunofluorescence localization in developing Torpedo marmorata electrocytes. In 40-45 mm Torpedo embryos, a stage of development at which the electrocytes exhibit a definite structural polarity, dystrophin, alpha/beta-dystroglycan and agrin accumulated concomitantly with acetylcholine receptors at the ventral pole of the cells. Among these components, agrin appeared as the most intensely concentrated and sharply localized. The scarcity of the nerve-electrocyte synaptic contacts at this stage of development, monitored by antibodies against synaptic vesicles, further indicates that before innervation, the machinery for acetylcholine receptor clustering is provided by electrocyte-derived agrin rather than by neural agrin. These observations suggest a two-step process of acetylcholine receptor clustering involving: (i) an instructive role of electrocyte-derived agrin in the formation of a dystrophin-based membrane scaffold upon which acetylcholine receptor molecules would accumulate according to a diffusion trap model; and (ii) a maturation and/or stabilization step controlled by neural agrin. In the light of these data, the existence of more than one agrin receptor is postulated to account for the action of agrin variants at different stages of the differentiation of the postsynaptic membrane in Torpedo electrocytes.

Agrin↗
PubMed1996

Anti-agrin staining is absent at abandoned synaptic sites of frog neuromuscular junctions.

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs, must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and alpha-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction.

Acetylcholinesterase↗
PubMed2000

Regulation of agrin expression in hippocampal neurons by cell contact and electrical activity.

Most synapses contain high concentrations of neurotransmitter receptors in the postsynaptic plasma membrane. Agrin (Ag) is an extracellular matrix protein necessary for the localization of acetylcholine receptors at the neuromuscular junction and for the differentiation of synapses in hippocampal neurons in vitro. The temporal pattern of agrin expression during the development of the central nervous system (CNS) is consistent with the notion that agrin expression is regulated during synaptogenesis. To identify the processes underlying this regulation, we have analyzed levels and alternative splicing of agrin mRNA in primary hippocampal neurons. Our results indicate that in the initial phases of synapse formation, contact-mediated processes and action potential-dependent neurotransmission regulate agrin mRNA expression, while secreted factors from glial cells, but not from hippocampal neurons, influence the alternative splicing of agrin mRNA. Previous studies have shown that specific agrin isoforms are able to induce the activation of a transcription factor and that secreted agrin associates with cellular surfaces. Therefore, we have tested whether agrin isoforms contribute to the contact-mediated induction of agrin expression in hippocampal neurons. None of the agrin isoforms tested in this study revealed this activity. Finally, we show that the role of evoked neural transmission in controlling agrin transcription changes during differentiation in vitro.

Action Potentials↗
PubMedDate not supplied

Agrin and microvascular damage in Alzheimer's disease.

Heparan sulfate proteoglycans (HSPGs) are ubiquitously present within the perivascular basement membrane, and have been shown to be altered in patients with Alzheimer's Disease (AD). Although the HSPG agrin clearly orchestrates the differentiation of the neuromuscular junction, its role in the brain remains unclear. Growing evidence suggests that agrin may be an important vascular basement membrane (VBM)-associated HSPG. In previous studies, we demonstrated that agrin is present throughout the brain microvasculature, as well as in neuronal cell bodies. AD brains exhibited fragmentation of VBM-associated agrin. Agrin immunoreactivity was also seen within senile plaques and neurofibrillary tangles. These changes were accompanied by the appearance of an additional pool of insoluble agrin. In the present study, we provide further evidence for microvascular damage in AD, by examining the distribution of agrin and laminin within the VBM, and by measuring the agrin concentration within hippocampus and prefrontal cortex. Furthermore, we assessed blood-brain-barrier (BBB) leakage by examining the perivascular distribution of prothrombin immunoreactivity. Soluble agrin levels were increased approximately 30% in Braak stage III-VI AD patients relative to age-matched controls. Furthermore, agrin and laminin exhibited identical patterns of VBM fragmentation in AD and colocalized with beta-amyloid in senile plaques. Microvascular changes were associated with the appearance of perivascular prothrombin immunoreactivity. Our data suggest that agrin is an important VBM-associated HSPG in the brain and that agrin levels are altered in association with microvascular damage in AD.

Aged↗
PubMed1997

Expression of agrin in the developing and adult rat brain.

Agrin, a synaptic basal lamina protein, is essential for the formation of the vertebrate neuromuscular junction. Agrin's role in synaptogenesis in the central nervous system has, however, not been elucidated. Therefore, we performed immunohistochemical analysis of agrin localization in adult rat brain using agrin-specific polyclonal antibodies. Our results show that agrin immunoreactivity is detected in neuronal cells throughout the brain, and that agrin is expressed in many morphologically and neurochemically distinct neuronal populations. Within neurons, agrin-immunoreactive material is present in dendrites. To determine agrin isoform expression in the central nervous system, we analysed the pattern of expression of several isoforms during development of the rat brain. Our results indicate that alternative splicing of agrin is specifically regulated in the nervous system; isoforms of the Y=4 (i.e. Ag x,4,0, Ag x,4,8 and Ag x,4,19), Z=8 and Z=19 type are expressed exclusively in the nervous system. Agrin expression precedes synaptogenesis and is developmentally regulated in neural tissues. To evaluate stimuli that may be involved in the regulation of agrin expression, we monitored the patterns of isoform expression following a depolarizing stimulus. Our results show that agrin expression in the adult hippocampus is regulated in an activity-dependent manner, with kinetics of induction resembling a delayed early response gene.

Aging↗
PubMed2002

Mapping sites responsible for interactions of agrin with neurons.

The multidomain proteoglycan agrin is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Agrin is also abundant in the brain, but its roles there are unknown. As a step toward understanding these roles, we mapped sites responsible for interactions of neurons with agrin. First, we used a series of recombinant agrin fragments to show that at least four sites on agrin interact with chick ciliary neurons. Use of blocking antibodies and peptides indicated that neurons adhere to a site in the second of three G domains by means of alphaVbeta1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct beta1 integrin. A third, integrin-independent adhesion site is near to but distinct from the site that induces postsynaptic differentiation in muscles. These domains are insufficient, however, to account for neurite outgrowth-inhibiting properties of full-length agrin, which are mediated by the N-terminal half of the molecule. We then used a second set of agrin mutants to demonstrate and map a transmembrane domain in the amino-terminus of the SN-isoform of agrin. The extracellular matrix-bound form of agrin, called LN, bears an amino-terminus required for secretion and binding to laminin. The SN form, which is selectively expressed by neurons, bears a variant amino terminus that converts agrin from a secreted, matrix-associated protein to a type-II transmembrane protein, providing a mechanism for presenting agrin in central, as opposed to neuromuscular, synaptic clefts. The SN-amino terminus can mediate externalization and membrane anchoring of heterologous proteins, but is insufficient to target them to the synapse. Together, these studies define sites that contribute to the subcellular localization of and signaling by neuronal agrin.

Agrin↗
PubMed1995

Agrin is a heparan sulfate proteoglycan.

In the present study we have identified the extracellular matrix protein agrin as a major heparan sulfate proteoglycan (HSPG) in embryonic chick brain. Using monoclonal antibodies and a polyclonal antiserum to the core protein of a previously identified HSPG from embryonic chick brain, our expression screened a random-primed E9 chick brain cDNA library. Twelve cDNAs were isolated that were shown to be identical to the chick extracellular matrix protein agrin. Western blot analysis and immunocytochemistry confirmed that agrin is a HSPG that is identical with the HSPG from embryonic chick brain. A polyclonal antiserum to recombinant agrin protein recognized agrin as a diffuse band of over 400 kDa in extracts from brain and vitreous humor. The agrin immunoreactivity on the blot was shifted to a defined band of approximately 250 kDa after treatment of the samples with heparitinase or nitrous acid, and this banding pattern was indistinguishable from immunoreactivity obtained with antibodies to the brain HSPG. We also show that agrin binds tightly to anion exchange beads, indicating that the molecule is highly negatively charged, which is a hallmark of all proteoglycans. Furthermore, the agrin antiserum recognizes the affinity purified HSPG from chick brain and vitreous humor. Immunocytochemistry demonstrated that agrin is expressed in developing brain, and is especially abundant in developing axonal tracts, in a distribution identical to the staining of the brain HSPG with monoclonal antibodies. We also show that the anti-HSPG antibodies stain the synaptic site of the neuromuscular junction, in agreement with agrin expression. Thus, our studies demonstrate that chick agrin is a HSPG that is prominent in the embryonic chick brain. Since previous studies from our laboratories have shown that this proteoglycan interacts with neural cell adhesion molecule, our studies raise the interesting possibility that neural cell adhesion molecule and agrin are interactive partners that may regulate a variety of cell adhesion processes during neural development, including synaptogenesis.

Agrin↗
PubMed1997

Selective regulation of agrin mRNA induction and alternative splicing in PC12 cells by Ras-dependent actions of nerve growth factor.

The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agriny0z0), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agriny4z8, agriny4z11, and agriny4z19) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agriny4 and agrinz8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.

Agrin↗