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:
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...
Abstract. Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (<4 ~tm 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to •15 ttm 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca ++. Co ++ and Mn ÷÷ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg +÷ and Sr ÷+ could not substitute for Ca ÷+. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.
The aims of the studies reported here were to determine the extent to which the specializations induced by agrin on cultured chick myotubes resemble the postsynaptic apparatus and examine how these specializations form. We found that agrin induces the formation of specializations at which at least 6 components of the postsynaptic apparatus are concentrated: one cytoplasmic component [a 43 kDa acetylcholine receptor (AChR)-associated protein], 3 membrane components [AChRs and globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)], and 2 extracellular matrix-associated proteins (A12 asymmetric AChE and a heparan sulfate proteoglycan). The accumulation of AChE and BuChE into agrin-induced aggregates occurred in the absence of any change in the amount, rate of synthesis, accumulation, and release, or molecular forms of either enzyme. Thus, agrin affects primarily the distribution of these components of the postsynaptic apparatus and not their metabolism. Agrin-induced formation of AChR aggregates was not prevented by inhibition of protein synthesis, consistent with our previous results that agrin-induced accumulation of AChRs occurs by lateral migration. The accumulation of components of the extracellular matrix would seem less likely to occur by lateral migration and so might require release of newly synthesized proteins; indeed, formation of aggregates of heparan sulfate proteoglycan was prevented by inhibitors of protein synthesis. Thus, different components of the postsynaptic apparatus accumulate in agrin-induced specializations by different mechanisms.
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