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

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.

Activation of Muscle-specific Receptor Tyrosine Kinase and Binding to Dystroglycan Are Regulated by Alternative mRNA Splicing of Agrin

“…Whereas the double mutant C45 NEI-B8-AES and the triple mutant C45 NEI-B8-AAA had a similarly low activity as the C45 B8 -8A mutant (Fig. 2C), the double mutant C45 NEI-B8-AAI had an EC 50 that was between wild-type C45 A4B8 and the C45 B8 -8A mutant. To see whether the NEImotif was the only critical determinant of agrin activity, we also tested the construct C45 HLSNEIPA-B8-AAANEIAA , where only the NEI motif is maintained and all the remaining amino acids in Mutation of all amino acids to alanine (C45 B8 -8A ) lowers potency strongly but does not abrogate all of the activity.…”

“…This result is consistent with the idea that ␣-dystroglycan can play an auxiliary role in MuSK phosphorylation. If ␣-dystroglycan would have an auxiliary role, inhibition of this binding should increase the apparent EC 50 of agrin in MuSK phosphorylation assays. We therefore measured MuSK phosphorylation in the presence of the agrin fragment N25C95 A0B0 (also called miniagrin), which binds to ␣-dystroglycan with high affinity (14).…”

“…The first set of agrin mutants was derived from a construct encoding a 45-kDa, C-terminal fragment (called C45 A0/4B0/8/11 ) that comprises LG2 and LG3 and one EGF-like (EG) domain. This fragment is known to be the smallest one that induces AChR aggregation with a half-maximal response (EC 50 ) at picomolar concentration (29). To test the influence of individual splice inserts independent of the second site, we also generated constructs encoding only LG2 or LG3 (Fig.…”

“…Thus, we next examined the influence of double and triple mutations. A conspicuous sequence of the B insert is the conserved tripeptide motif Asn-Glu-Ile (NEI) because the first two amino acids could complex Ca 2ϩ , which has been shown to be important for the agrin AChR clustering activity (50,51). Indeed, double or triple mutations of the NEI motif affected the agrin activity strongly.…”

Wnt signaling promotes AChR aggregation at the neuromuscular synapse in collaboration with agrin

“…At the vertebrate cholinergic neuromuscular junction (NMJ), agrin, a heparan sulfate proteoglycan secreted by motoneurons (6,7), induces post-synaptic differentiation by aggregating acetylcholine receptors (AChR) and other proteins at the post-synaptic membrane (8)(9)(10). This effect is mediated through sequential activation of Rho GTPases; agrin induces a rapid and transient activation of Rac1 that is necessary for the initial phase of AChR micro-cluster formation, whereas the subsequent RhoA activation is crucial for the coalescence of the micro-clusters into full-sized AChR clusters (11,12).…”