A hallmark of neuromuscular junction development is the formation of high-density acetylcholine receptor (AChR) clusters at the postsynaptic muscle membrane. In cultured muscle cells, AChRs spontaneously form clusters (pre-patterned clusters) in the absence of synaptogenic stimuli. Upon motor innervation, the nerve-derived factor agrin activates muscle-specific kinase MuSK to disperse AChRs from pre-patterned clusters and to induce new clusters at postsynaptic sites. In this study the roles of tyrosine phosphatases (PTPs) in AChR dispersal and clustering were investigated....[ Read more ]

A hallmark of neuromuscular junction development is the formation of high-density acetylcholine receptor (AChR) clusters at the postsynaptic muscle membrane. In cultured muscle cells, AChRs spontaneously form clusters (pre-patterned clusters) in the absence of synaptogenic stimuli. Upon motor innervation, the nerve-derived factor agrin activates muscle-specific kinase MuSK to disperse AChRs from pre-patterned clusters and to induce new clusters at postsynaptic sites. In this study the roles of tyrosine phosphatases (PTPs) in AChR dispersal and clustering were investigated.

It was found that HB-GAM beads, which mimic motor innervation, induce AChR clustering in cultured Xenopus muscle cells and disperse pre-patterned AChR clusters. But, following PTP inhibition, AChRs reform clusters at the original sites of dispersed pre-patterned clusters. This suggests that the scaffolding structure of pre-patterned cluster is capable of re-recruiting AChRs after NMJ is established and PTPs suppress the formation of extrasynaptic AChR clusters. Furthermore, it was found that PTP inhibition reduces surface AChR mobility, suggesting that PTPs regulate AChR redistribution by mobilizing AChRs on muscle cell surface.

To further understand the molecular nature of the PTP regulation in the formation of AChR clusters, a non-receptor PTP Shp2 and its activating molecule SIRPα1 were examined. Suppression of Shp2 activity by either pharmacological inhibitor or dominant negative Shp2 promotes the formation of both pre-patterned and agrin-induced AChR clusters in C2C12 myotubes. Introduction of constitutively active Shp2 protein has opposite effects. Overexpression of SIRPα1, a Shp2 activating protein which is tyrosine-phosphorylated upon agrin treatment, decreases both pre-patterned and agrin-induced AChR clusters whereas dominant negative SIRPα1 causes an increase in cluster formation. Moreover, co-expression of dominant negative SIRPα1 with active Shp2 or wild-type SIRPα1 with inactive Shp2 reverses the effects of exogenous Shp2 proteins.

By using Xenopus primary muscle culture, the specific roles of Shp2 in AChR cluster dispersal were revealed. Shp2 inhibitor promotes the formation of AChR microclusters and suppresses HB-GAM bead-induced dephosphorylation and dispersal of pre-patterned AChR clusters. The expression of wild-type or active Shp2 or Shp2-activating full-length SIRPα1 promotes AChR cluster dispersal induced by beads while truncated SIRPα1 which is unable to activate Shp2 decreases dispersal. The same results were obtained when muscle cells were stimulated with motoneurons. Taking together, Shp2, which is activated by molecules like SIRPα1 downstream of MuSK, balances the action of tyrosine kinases in AChR cluster formation and mediates AChR cluster dispersal.

As the pool of diffuse surface AChRs has a direct bearing on cluster formation and dispersal, their endocytosis was also examined with the aid of quantum dots. Dynasore, a specific dynamin GTPase inhibitor, blocks AChR internalization induced by cross-linking, indicating the dynamin-dependence of this process. Dynamin inhibition also suppresses HB-GAM bead-induced AChR cluster dispersal, suggesting that internalization contributes to AChR removal from aneural clusters. Moreover, AChR internalization is suppressed by PTP inhibitor pervanadate but not by a Shp2-specific inhibitor NSC. These data suggest that PTP is also involved in AChR endocytosis. However, different PTPs are probably used for AChR cluster dispersal versus endocytosis.