Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.

Abstract

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers . Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization . Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate . We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction.The insoluble fraction from the electric organ was rich in extracellular matrix constituents ; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils . It caused a 3-to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes . The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChRaggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied . Activity was destroyed by heat and by trypsin . The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5 .5 buffers . The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component .Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5 .5 extracts from the muscle fraction . The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction . This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath . Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina .Each muscle fiber in skeletal muscles is ensheathed by basal lamina, a component ofextracellular matrix that lies adjacent to the myofiber plasma membrane. The portion of the basal THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 615-627 C The Rockefeller University Press -0021-9525/84/08/0615/13 $1 .00 lamina in the synaptic cleft at the neuromuscular junction is h...

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…One synaptic molecule implicated in the activation of a tyrosine kinase pathway is agrin. This nerve-derived protein originally was isolated for its striking ability to cause AChR aggregation in chick myotubes (Godfrey et al, 1984). The observation that agrin induces tyrosine phosphorylation of the ␤ (Wallace et al, 1991;Qu and Huganir, 1994;Wallace, 1994;Ferns et al, 1996), and perhaps the ␦ (Qu and Huganir, 1994), subunit of the AChR has led to the hypothesis that this is a critical, and perhaps a prerequisite, step in the AChR clustering pathway.…”

“…Postsynaptic membranes of this tissue have provided a rich source for the identification and characterization of many synaptic proteins, including rapsyn, syntrophin, and agrin (Sobel et al, 1977;Godfrey et al, 1984;Froehner et al, 1987). To identify potential binding partners for Grb2 in Torpedo postsynaptic membranes, we used a protein overlay assay in which GST fusion proteins were used to probe membrane proteins separated by SDS-PAGE and transferred to nitrocellulose.…”

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

“…The chimeric constructs were transfected into COS-7 cells, and the conditioned medium was tested after 2 days for its ability to induce AChR aggregation on cultured chick myotubes under the standard conditions for assaying agrin's activity (Godfrey et al, 1984;Wall ace, 1986). The exchange of the Kpnl fragment (Figure 1) between CBA-1 and ARP-2 cDNA , the shorter of the two agrin-related cDNAs, resulted in two chimeric DNAs_ One having the same 5' end as CBA-1 but lacking regions A and B encoded an CBA-1…”

“…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.…”

cDNA that encodes active agrin

“…The active protein in the medium conditioned by COS-7 cells transfected with CBA-1 induced the formation of AChR aggregates in a dose-dependent way, as does T. californica agrin (Godfrey et al, 1984). Moreover, the maximal number of aggregates induced by conditioned medium was not increased by the simultaneous addition of T. californica agrin to myotube cultures, indicating that the secreted protein ac ted by the same mechanisms as those used by agrin (conditioned medium, 16.3 ± 0.7 AChR aggregates per myotube segment; conditioned medium + 2 U ofT.…”