Intracellular calcium regulates agrin-induced acetylcholine receptor clustering

Abstract

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.

Fig. 1.

The number of spontaneous and agrin-induced AChR clusters is decreased in BAPTA-loaded cells. A, Myotubes were incubated with 50 μm BAPTA-AM (bottom panels) or vehicle only (top panels), washed, and incubated with or without agrin for 4 hr, as indicated. Cultures were then incubated in rhodamine-α-BTx and examined by fluorescence microscopy to reveal the distribution of AChRs. B, Quantitation of AChR clusters revealed that significantly fewer spontaneous and agrin-induced clusters are observed in BAPTA-loaded cells. AChR clusters were quantitated as described in Materials and Methods. Values are mean ± SEM averaged from seven separate experiments. *p < 0.05, paired Student’st test. Scale bar, 20 μm.

Fig. 2.

Quantitation of agrin-induced and spontaneous AChR clusters in myotubes loaded with varying concentrations of BAPTA-AM. Myotubes were incubated with the indicated concentrations of BAPTA-AM for 1 hr and then incubated in media with or without agrin for 4 hr. Data shown are from one representative experiment and are expressed as mean ± SEM. Similar results were seen in three additional experiments.

Fig. 3.

Inhibition of AChR clustering in BAPTA-loaded cells is reversible. Cells were loaded with 50 μmBAPTA-AM or vehicle for 1 hr, washed, and then incubated with agrin either immediately or 24 hr later (Washout). After the wash-out period, the numbers of spontaneous and agrin-induced AChR clusters returned to levels similar to vehicle-treated cells. Values are expressed as mean ± SEM from one experiment. Similar results were observed in four other experiments.

Fig. 4.

Antibody-driven AChR microclustering is unaffected in BAPTA-loaded cells. Myotubes were incubated with vehicle only (A, B) or 50 μm BAPTA-AM (C) and then directly incubated in buffer alone (A) or anti-AChR antibody mAb 35 and anti-Rat IgG for 1 hr at 37°C (B, C). The distribution of AChRs was then determined by labeling with rhodamine-α-BTx. In the absence of anti-AChR antibody incubation, AChRs were distributed diffusely on the myotube surface (A). Incubation with anti-AChR antibodies caused extensive AChR microclustering in both vehicle (B) and BAPTA-loaded (C) cells. Scale bar, 20 μm.

Fig. 5.

Spontaneous and agrin-induced AChR clusters in EGTA-loaded cells. Myotubes were loaded with EGTA by incubating them with 50 μm EGTA-AM for 1 hr at 37°C and then incubating them with or without agrin, as in Figure 1. The numbers of neither spontaneous nor agrin-induced AChR clusters were significantly different in EGTA-loaded, as compared with vehicle-loaded cells. Mean ± SEM from one representative experiment;p = 0.392 and 0.360 for spontaneous and agrin-induced clusters, respectively, Student’s t test. Similar results were obtained by using cells loaded with 25 or 100 μm EGTA-AM.

Fig. 6.

Agrin-induced tyrosine phosphorylation of AChR β-subunit in BAPTA-loaded cells. Myotubes were loaded with BAPTA or vehicle and then incubated with or without agrin for 4 hr, as indicated. Surface AChRs were affinity-purified with biotinylated α-BTx and separated by SDS-PAGE. A, Immunoblots were probed with mAb 4G10 to visualize tyrosine phosphorylated proteins (p-tyr) or mAb 61 to visualize the AChR α-subunit (anti-α). In parallel blots, mAb 124 was used to identify the AChR β-subunit (data not shown). Agrin induced tyrosine phosphorylation of AChR β-subunit in both the presence and absence of BAPTA. A second polypeptide of slightly slower mobility was phosphorylated also. This polypeptide was identified tentatively as AChR δ-subunit, on the basis of immunoreactivity with mAb 88b (data not shown) and previous reports showing that agrin also induces the phosphorylation of this subunit (Qu and Huganir, 1994).B, Phosphotyrosine levels of the AChR β-subunit were measured and expressed relative to total AChR levels, as described in Materials and Methods. Results were derived from three separate experiments, each normalized to untreated controls. Agrin-induced AChR β-subunit tyrosine phosphorylation was equivalent in vehicle, as compared with BAPTA-loaded cells. The basal level of AChR β-subunit tyrosine phosphorylation was also not significantly different in BAPTA-loaded cells (p > 0.05; Newman–Keuls multiple comparison test, after repeated measures ANOVA of three separate experiments; data not normalized).

Fig. 7.

Two models of the signaling pathway of agrin. The first step in the signaling pathway is the activation of the MuSK receptor tyrosine kinase by agrin. The subsequent tyrosine phosphorylation of the AChR is dependent on MuSK activation. The results presented here are consistent with two models. (1) The intracellular calcium-regulated step occurs downstream of AChR phosphorylation. (2) The AChR phosphorylation is on a pathway parallel to (but not necessarily required for) agrin-induced AChR clustering. See Discussion for details.