Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase.

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin-induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin-induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester.

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 (less than 4 micron 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 approximately 15 micron 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.

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes.

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.

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

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 two peaks of AChR/AChE/BuChE-aggregation 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."

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

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 AChR-aggregating 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.

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.

Selective loss of neurites during differentiation of cells in the leech central nervous system.

The arborizations of annulus erector (AE) motoneurons in the central nervous system of the leech, Hirudo medicinalis, have been examined during embryogenesis to determine how segmental differences in their branching patterns arise. Early in development AE motoneurons all along the ganglionic chain had a similar central arborization, with major branches extending both rostrally and caudally along the connectives that link adjacent ganglia. As the embryo grew, the processes in the connectives elongated but failed to increase significantly in caliber and eventually atrophied and were lost. This sequence of events did not occur uniformly along the cord, however. AE motoneurons in midbody ganglia lost both anterior and posterior branches, cells near the head lost only their posterior branch, while cells near the tail lost only their anterior branch. In this way, the selective atrophy of neurites during development produced a systematic segmental difference in the morphology of homologous cells.

Segmental variation in the arborization of identified neurons in the leech central nervous system.

Mechanosensory and motor neurons in the central nervous system of the leech have been examined by intracellular injection of horseradish peroxidase and electrophysiological mapping of their peripheral fields to determine how the arborizations of homologous cells are influenced by their segmental position. The branching patterns of annulus erector (AE) motoneurons in ganglia near the head and tail were found to be more extensive than those of cells in midbody ganglia. As in midbody ganglia, the peripheral fields of AE motoneurons in adjacent ganglia near the head and tail overlapped extensively, but the subfields innervated by individual branches of a single AE motoneuron showed little or no overlap. No AE motoneurons were found in the head ganglion or in the 20th and 21st free segmental ganglia. The branching pattern of touch-sensitive mechanosensory cells showed a similar segmental variation; touch cells in ganglia near the head and tail had more extensive arborizations than those in midbody ganglia. The rostrocaudal position along the cord at which the branching pattern changed from that characteristic of midbody ganglia to one with a more extensive arborization differed for different types of neurons. These findings demonstrate that a cell's pattern of arborization is not determined by a simple segmental difference between ganglia and suggest that during development neurons respond individually to cues that vary along the length of the cord.

Distribution of AChE in cholinergic and non-cholinergic neurons.

Intracellular and surface acetylcholinesterase activities were determined for individual cholinergic and non-cholinergic neurons dissected from the central nervous system of the leech. Echothiophate pretreatment was used to inhibit selectively extracellular enzyme. Cells releasing acetylcholine as a transmitters had approximately 10-fold higher levels of intracellular acetylcholinesterase activity, while all neurons had similar levels of activity associated with the cell surface. These results suggest that intracellular cholinesterase may be a useful marker for cholinergic neurons.

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.

Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components 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.

Signaling mechanisms mediating synapse formation.

Recent experiments have begun to decipher the molecular dialog that mediates differentiation at sites of synaptic contact between neurons and their targets. It had been hypothesized that the protein agrin is released by axon terminals at embryonic neuromuscular junctions and binds to a receptor on the myofiber surface to trigger postsynaptic differentiation. Now a genetic 'knockout' experiment has confirmed the essential role of agrin in signaling between developing nerve and muscle. A second 'knockout' has shown that the muscle-specific receptor tyrosine kinase MuSK is a critical element in the agrin-induced signaling cascade. Additional results suggest that MuSK may comprise a portion of the agrin receptor.

The mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin, a protein isolated from the synapse-rich electric organ of Torpedo californica, induces the formation of specializations on myotubes in culture which resemble the post-synaptic apparatus at the vertebrate skeletal neuromuscular junction. For example, the specializations contain aggregates of acetylcholine receptors and acetylcholinesterase. This report summarizes the evidence that the formation of the post-synaptic apparatus at developing and regenerating neuromuscular junctions is triggered by the release of agrin from motor axon terminals and describes results of recent experiments which suggest that agrin-induced tyrosine phosphorylation of the beta subunit of the acetylcholine receptor may play a role in receptor aggregation.

Inhibition of agrin-induced acetylcholine-receptor aggregation by heparin, heparan sulfate, and other polyanions.

Heparin and heparan sulfate have been shown to block nerve-induced acetylcholine-receptor (AChR) aggregation at developing neuromuscular junctions. We found that heparin, heparan sulfate, and a wide variety of other polyanions also inhibited agrin-induced AChR aggregation. The more highly charged the polyanion, the more potent it was as an inhibitor. Inhibition of agrin-induced AChR aggregation was due, at least in part, to the formation of a complex between the polyanion and agrin that was inactive. These findings are consistent with the hypothesis that nerve-induced aggregation of AChRs is mediated by the release of agrin, or a closely related protein, from axon terminals and suggest that a polyanion, such as a sulfated proteoglycan, may be involved in the interaction of agrin with its receptor on the myotube surface.

Formation of the neuromuscular junction: molecules and mechanisms.

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron-neuron synapses in the peripheral as well as the central nervous system.

Characterization of acetylcholinesterase in individual neurons in the leech central nervous system.

Acetylcholinesterase (AChE) activity was measured in cholinergic and non-cholinergic neurons in the central nervous system of the leech. Intracellular AChE was assayed by pretreating intact ganglia with echothiophate to inhibit selectively extracellular enzyme. The concentration of intracellular AChE in cholinergic neurons was 3- to 24-fold higher than that in non-cholinergic cells. The properties of AChE in extracts of leech ganglia were similar to those of "true" acetylcholinesterase, although butyrylthiocholine was almost as good a substrate as acetylthiocholine. There was also cholinesterase activity in leech blood; this enzyme resembled butyrylcholinesterase. Sucrose gradient velocity sedimentation of Triton X-100 extracts of leech ganglia revealed a major peak of AChE activity at 6.5 S and a small peak at 4.3 S. The pattern of activity in the gradient was the same when intact ganglia were pretreated with echothiophate, although the total activity was reduced by 98%. Intact leech ganglia were stained for AChE activity with and without echothiophate pretreatment. In ganglia that had not been exposed to echothiophate, cholinesterase reaction product was deposited primarily on the ganglionic sheath. In pretreated ganglia, on the other hand, cholinesterase activity was concentrated within neuronal cell bodies. Electrophysiological identification and intracellular injection of the fluorescent dye Lucifer Yellow prior to staining were used to confirm that most AChE-positive cells were cholinergic motoneurons. Two previously unidentified neurons staining for AChE were shown to be motoneurons. These results demonstrate that cholinergic motoneurons can be differentiated from other cells in the leech nervous system by their high intracellular concentration of AChE.