Abstract
Agrin is a basement membrane protein crucial for development and maintenance of the neuromuscular junction in vertebrates. The C. elegans genome harbors a putative agrin gene agr-1. We have cloned the corresponding cDNA to determine the primary structure of the protein and expressed its recombinant fragments to raise specific antibodies. The domain organization of AGR-1 is very similar to the vertebrate orthologues. C. elegans agrin contains a signal sequence for secretion, seven follistatin domains, three EGF-like repeats and two laminin G domains. AGR-1 loss of function mutants did not exhibit any overt phenotypes and did not acquire resistance to the acetylcholine receptor agonist levamisole. Furthermore, crossing them with various mutants for components of the dystrophin-glycoprotein complex with impaired muscle function did not lead to an aggravation of the phenotypes. Promoter-GFP translational fusion as well as immunostaining of worms revealed expression of agrin in buccal epithelium and the protein deposition in the basal lamina of the pharynx. Furthermore, dorsal and ventral IL1 head neurons and distal tip cells of the gonad arms are sources of agrin production, but no expression was detectable in body muscles or in the motoneurons innervating them. Recombinant worm AGR-1 fragment is able to cluster vertebrate dystroglycan in cultured cells, implying a conservation of this interaction, but since neither of these proteins is expressed in muscle of C. elegans, this interaction may be required in different tissues. The connections between muscle cells and the basement membrane, as well as neuromuscular junctions, are structurally distinct between vertebrates and nematodes.
Highlights
Agrin is a large proteoglycan with a prominent function at the developing neuromuscular junction (NMJ) where it plays a pivotal role in the formation and maintenance of the acetylcholine receptor (AChR) clusters
Further studies revealed that agrin is synthesized by motor neurons that release it into the synaptic cleft where it stably integrates into the synaptic basal lamina (BL), a specialized thin layer of the extracellular matrix (ECM) [3,4,5]
In WormBase, the online database of the C. elegans genome, the agrin gene was mapped to the cosmid F41G3, originally as two separate open reading frames (ORFs) named F41G3.12 and F41G3.15, corresponding to the 59 and the other to the 39 part of vertebrate agrin, respectively
Summary
Agrin is a large proteoglycan with a prominent function at the developing neuromuscular junction (NMJ) where it plays a pivotal role in the formation and maintenance of the acetylcholine receptor (AChR) clusters. Further studies revealed that agrin is synthesized by motor neurons that release it into the synaptic cleft where it stably integrates into the synaptic basal lamina (BL), a specialized thin layer of the extracellular matrix (ECM) [3,4,5]. Based on these findings, McMahan proposed the ‘agrin hypothesis’ which states that agrin is a nerve-derived synaptic organizing molecule [6] (reviewed in [7,8]). Isoforms expressed by motoneurons, which contain inserts at the B/z splice site, are active in AChR clustering, whereas agrin expressed by muscle lacks the inserts and does not cluster AChRs
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