Identification of agrin, a synaptic organizing protein from Torpedo electric organ.

Abstract

Abstract. Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (ACHE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed tw~ peaks of AChR/ AChE/BuChE-aggregating activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135-and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin:

Cerebral Ischemia Causes Dysregulation of Synaptic Adhesion in Mouse Synaptosomes

“…Agrn is an essential component of the basal lamina within the neuromuscular junction, where its organizational role is well understood (Nitkin et al, 1987). We detected Agrn in our synaptosomal preparations confirming its presence in the central nervous system synapse.…”

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution

“…Native agri n has been best characterized in basal lamina extracts of the electric o rgan of the ray T. californica. The extracts contain two forms of agrin, 150 and 95 kd, both of which have AChR/AChE aggregating activity (Nitkin et al, 1987). Both proteins are likely to be derived from a larger protein that is proteolytically cleaved either during tissue extraction or during in situ posttranslational modification; the 95 kd form is located in the C-terminal half of the large protein (Smith et al, submitted; Tsim et al, 1992).…”

“…After overnight incubation at 4°C, 100 !ll of a 1:1 suspension of protein A-Sepharose beads (Pharmacia) was added, and the incubation was continued for 2 hr at room temperature. Beads were pelleted by centrifugation and washed sequentially as described in Nitkin et al (1987). Bound antigen-antibody complexes were released by boiling for 5 min in 60 !ll of SDS-polyacrylamide gel electrophoresis sam pie buffer (3% SDS, 5% 2-mercaptoethanol, 15% glycerol, 0.001% brom phenol blue in 62.5 mM Tris-HCI [pH 6.5]).…”

“…However, basal lamina at nonsynaptic sites in a variety of tissues including muscle, central nervous system (CNS), heart, and kidney, where AChRs and AChE are not known to aggregate, is also stained by these antibodies (Reist et al, 1987;Godfrey et al, 1988aGodfrey et al, , 1988b. Moreover, proteins inactive in AChR aggregation assays are immunoprecipitated from extracts of several different tissues by antiagrin MAbs (Nitkin et al, 1987;Godfrey, 1991). Such findings have led to the suggestion that anti-agrin MAbs also recognize proteins other than agrin and that these proteins differ from agrin in their function.…”

“…Transfection experiments revealed that the proteins encoded by these cDNAs are recognized by anti-agrin antibodies, but unlike agrin, they are inactive in standard AChR aggregation assays. Because the discovery of agrin was based on its ability to cause AChRs to aggregate in cultured chick myotubes (Godfrey et al, 1984;Nitkin et al, 1987) and because the two proteins we describe here lack the AChR aggregating activity, but are highly similarto agrin in structure, we refer to them simply as agrin-related proteins 1 and 2 (ARP-1 and ARP-2). The predicted amino acid sequences of the agrin-related proteins are identical to that of agrin except for the absence of a stretch of 11 amino acids in ARP-1 and stretch es of the 11 and 4 amino acids in ARP-2.…”