Evidence of an Agrin Receptor in Cortical Neurons

Abstract

Agrin plays a key role in directing the differentiation of the vertebrate neuromuscular junction. Understanding agrin function at the neuromuscular junction has come via molecular genetic analyses of agrin as well as identification of its receptor and associated signal transduction pathways. Agrin is also expressed by many populations of neurons in brain, but its role remains unknown. Here we show, in cultured cortical neurons, that agrin induces expression of the immediate early gene c-fos in a concentration-dependent and saturable manner, as expected for a signal transduction pathway activated by a cell surface receptor. Agrin is active in cortical neurons at picomolar concentrations, is Ca 2ϩ dependent, and is inhibited by heparin and staurosporine. Despite marked differences in acetylcholine receptor (AChR)-clustering activity, all alternatively spliced forms of agrin are equally potent inducers of c-fos in cortical neurons. A similar, isoform-independent response to agrin was also observed in cultures prepared from the hippocampus and cerebellum. Only agrin with high AChR-clustering activity was effective in cultured muscle, whereas non-neuronal cells were agrin insensitive. Although consistent with a receptor tyrosine kinase model similar to the muscle-specific kinase-myotubeassociated specificity component complex in muscle, our data suggest that CNS neurons express a unique agrin receptor. Evidence that neuronal signal transduction is mediated via an increase in intracellular Ca 2ϩ means that agrin is well situated to influence important Ca 2ϩ -dependent functions in brain, including neuronal growth, differentiation, and adaptive changes in gene expression associated with synaptic remodeling.

Agrin regulates growth cone turning ofXenopusspinal motoneurons

“…Although there is ample evidence of an agrin-dependent signaling pathway in neurons (Hilgenberg et al, 1999;Ji et al, 1998;Karasewski and Ferreira, 2003), little is known about the identity of the neuronal agrin receptor. Recently, the domain of agrin that binds to its receptor in neurons was identified (Burgess et al, 2002;Hoover et al, 2003).…”

“…2A). To examine the specificity of agrin in inhibiting the extension of neurites, an agrin-neutralizing antibody (AGR-530) previously reported to block agrin-induced AChR clustering at the NMJ was used (Hilgenberg et al, 1999). Addition of this neutralizing antibody to the agrin solution used in the above neurite growth experiments completely abolished the inhibition effect of the agrin solution on neurite extension (Fig.…”

“…Treatment of primary neuronal cultures with agrin leads to the activation of cAMP-response element binding protein (Ji et al, 1998) and the expression of c-fos (Hilgenberg et al, 1999). To initiate its action on neurons, agrin is likely to first activate a cell-surface receptor.…”

Fast Synaptic Currents inDrosophilaMushroom Body Kenyon Cells Are Mediated by α-Bungarotoxin-Sensitive Nicotinic Acetylcholine Receptors and Picrotoxin-Sensitive GABA Receptors

“…After 24 hr, 0.5 ml of a 3:1 mixture of DDM2 and conditioned neurobasal medium (cNBM) was added to each dish. cNBM (neurobasal medium containing B27 supplements; Invitrogen, Gaithersburg, MD) was conditioned for 24 hr by non-neuronal mouse brain feeder cell cultures (Hilgenberg et al, 1999). Cultures were fed every 4 -5 d by removing 1 ml of media from the dish and adding 1 ml of the 3:1 mixture of DDM2 and cNBM.…”

HIV-1-infected Blood Mononuclear Cells Form an Integrin- and Agrin-dependent Viral Synapse to Induce Efficient HIV-1 Transcytosis across Epithelial Cell Monolayer

“…Different isoforms of agrin arise from alternative mRNA splicing. These include a transmembrane form and an isoform bound to the basement membrane (Hilgenberg et al, 1999;Neumann et al, 2001;Hoover et al, 2003). Agrin contains one heparin-and one integrin-binding site.…”