The mechanism of agrin-induced acetylcholine receptor aggregation

Abstract

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.

Src-Family Kinases Stabilize the Neuromuscular SynapseIn Vivovia Protein Interactions, Phosphorylation, and Cytoskeletal Linkage of Acetylcholine Receptors

“…Agrin treatment is known to strengthen the AChRcytoskeletal link in the process of cluster formation (Wallace, 1992(Wallace, , 1995Borges and Ferns, 2001;Moransard et al, 2003). This is manifested as a decrease of solubilized and precipitated AChR in the first extraction and an increase of receptor in the second extraction (Borges and Ferns, 2001;Moransard et al, 2003).…”

Lipid Rafts Serve as a Signaling Platform for Nicotinic Acetylcholine Receptor Clustering

“…However, agrin-induced Rac1 activation was inhibited in myotubes treated with either MCD or PDMP. The AChR becomes tyrosine phosphorylated after agrin stimulation (Wallace, 1991). Phosphorylation of the AChR is implicated in AChR clustering by regulating the association of the receptor with cytoskeletal proteins (Wallace, 1995;Borges and Ferns, 2001).…”

Synaptic differentiation is defective in mice lacking acetylcholine receptor β-subunit tyrosine phosphorylation

“…myotubes Agrin treatment slows the rate of AChR extraction with non-ionic detergent, suggesting that agrin stimulation leads to a more stable association of AChRs with the cytoskeleton (Wallace, 1992;Borges and Ferns, 2001;Moransard et al, 2003). To determine whether tyrosine phosphorylation of the AChR ␤-subunit is necessary to stabilize AChRs, we treated wild-type and AChR-␤ 3F/3F myotubes with agrin for 3 hours, labeled AChRs with 125 I-␣-BGT for 1 hour in the presence of agrin, extracted labeled AChRs with non-ionic detergent for several minutes and measured the rate of AChR extraction.…”