Mapping sites responsible for interactions of agrin with neurons

• Published in: Journal of neurochemistry Vol. 83; no. 2; pp. 271 - 284
• Main Authors: Burgess, Robert W., Dickman, Dion K., Nunez, Lorna, Glass, David J., Sanes, Joshua R.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-10-2002; Blackwell
• Subjects: acetylcholine receptor adhesion; Agrin - metabolism; Amino Acid Sequence; Animals; Binding Sites - physiology; Biological and medical sciences; Cell Adhesion - physiology; Cell interactions, adhesion; Cell Membrane - metabolism; Cells, Cultured; Chick Embryo; COS Cells; Fibroblasts - cytology; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; integrin; Integrins - metabolism; Molecular and cellular biology; Molecular Sequence Data; Neurons - cytology; Neurons - metabolism; Peptide Fragments - metabolism; Protein Binding - physiology; Protein Structure, Tertiary - physiology; Structure-Activity Relationship; synapse; transmembrane; Proteoglycan; Molecular structure; Cell cell interaction; Adhesion; Neuromuscular junction; Vertebrata; Cholinergic receptor; Synapse; Epidermal growth factor; Integrin; Neuron; Agrin; Extracellular matrix; Chicken; Aves; Differentiation
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2002.01102.x

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 αVβ1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct β1 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.