Cooperative Regulation by Rac and Rho of Agrin-induced Acetylcholine Receptor Clustering in Muscle Cells

Abstract

During embryonic development, innervation induces the anatomical and biochemical specialization of a defined region of the muscle cell membrane immediately under the motor nerve ending. A prominent aspect of this specialization is the accumulation of high densities of nicotinic acetylcholine receptors (AChR) 1 at these sites (1, 2). The aggregation of AChR and other synaptic components is mediated by agrin, a heparansulfated proteoglycan that is synthesized by motor neurons and secreted into the synaptic cleft (3, 4). The recruitment of AChR into clusters in postsynaptic membranes ensures high efficiency synaptic transmission at neuromuscular junctions.Agrin-induced redistribution of surface AChR involves the co-clustering of multiple associated proteins, several of which have been identified to date (2, 5). These include the musclespecific receptor tyrosine kinase (MuSK) (6), the linker protein rapsyn (7), and the scaffolding proteins dystroglycan and utrophin (8). The clustering of MuSK upon its activation by agrin, the formation of AChR complexes with rapsyn, as well as the aggregation of these complexes and their stabilization upon the formation of dystroglycan-utrophin scaffolds appear to be sequential events that are to some extent independently regulated (9 -11). Although the signaling mechanisms that couple agrin activation of MuSK to the clustering of postsynaptic components are incompletely characterized, there is recent evidence for the participation of Src tyrosine kinases (12, 13), the Rho GTPases Rac and Cdc42 (14), and Dishevelled, a component of the Wnt signaling pathway (15).Focal changes in the peripheral actin-based cytoskeleton are thought to underlie the aggregation of AChR at neuromuscular junctions (16 -18). The monomeric G proteins Rac and Rho function to link extracellular signals to dynamic changes in actin cytoskeleton organization leading to the assembly of lamellipodia and actin-myosin filaments, respectively (19 -22). Rac activation induces actin polymerization at the plasma membrane, causing the appearance of lamellipodia with resultant stimulation of cell spreading and motility (23,24). Rho exerts the opposite effect by stimulating actin stress fiber appearance and focal adhesion complex formation to promote cell adhesion and contractility (25,26). As several recent studies have shown, Rac and Rho are mutually inhibitory in several cell types, and the balance between their antagonistic activities is responsible for the dynamic changes in cell morphology, adhesion, and motility (27,28). In other systems Rac serves as an upstream activator of Rho (29).We have recently shown that agrin triggers the activation of Rac and Cdc42 and that this activation is necessary but not sufficient for formation of full size AChR clusters (14). In this study we present evidence that Rho plays a crucial role in agrin-initiated signaling that is complementary to the contribution of Rac/Cdc42 and that together these Rho family GTPases serve to couple signaling initiated by extracellular agrin to the for...

ATP Potentiates Agrin-induced AChR Aggregation in Cultured Myotubes

“…This result was in line with a previous report (12), except the elapsed time here is slightly shorter, which could be a result of the difference in myotube species. Weston et al (12) used C2C12 myotubes, and we used primary chick myotubes. In addition, this time course of RhoA activation was very similar for both types of assays in measuring RhoA activation.…”

“…Moreover, Rac is proposed to play a role in the formation of agrin-induced small AChR aggregates; RhoA is required, subsequently, to condense those small aggregates as large agrin-induced AChR aggregates. The small GTPases are known to induce the re-arrangement of intracellular cytoskeleton (40), which, therefore, could explain the clustering of AChRs in the post-synaptic muscle (12,41). Here, we demonstrate further the potentiation role of P2Y 1 receptor in agrininduced AChR aggregation could also involve the small GTPases.…”

“…The signaling mechanism of agrin-induced Rac and RhoA activation in cultured myotubes has been described previously (11,12). The agrin-induced Rac activation is rapid and transient, and it has been proposed to be a prerequisite for the activation of RhoA.…”

“…Several downstream effects of agrin and MuSK signaling have been proposed. Agrin has been shown to activate Rho family of guanosine triphosphatase (GTPase) Rac, Cdc-42, and RhoA, and these molecules are required for agrininduced AChR aggregation in myotubes (11,12). The mobilization of intracellular Ca 2ϩ in post-synaptic muscle has been demonstrated to regulate the formation and/or the maintenance of agrin-induced AChR aggregation (13)(14)(15).…”

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

“…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). Although initial evidence suggested that agrin was crucial for initiation of post-synaptic development (6,7), agrin also plays a later maintenance role (13,14).…”