br Results br Discussion Utilizing a proteomic

Results
Discussion Utilizing a proteomic approach, we identified HSP90β as a protein that becomes associated with surface AChR in agrin-stimulated muscle cells. We demonstrate that HSP90β does not interact with the AChR directly; instead, via direct interaction with rapsyn, HSP90β becomes associated with clustered AChR. Consistently, HSP90β is enriched at the NMJ and in AChR clusters in muscle cells. We show that HSP90β regulates AChR clustering. First, treatment of muscle AZD 3965 with 17-AAG, an HSP90 inhibitor that disrupts the AChR-HSP90β complex, attenuated agrin-induced AChR clustering. 17-AAG also reduced the stability of AChR clusters. Second, suppression of HSP90β expression inhibits agrin-induced AChR clustering in muscle cells. Third, disruption of the HSP90β-rapsyn interaction by a dominant-negative approach inhibits AChR clustering. Mouse embryos injected with 17-AAG form fewer AChR clusters, suggesting that HSP90β is involved in NMJ formation in vivo. Finally, we provide evidence that HSP90β regulates rapsyn stability. Rapsyn was reduced in muscle cells treated with 17-AAG, cells expressing HSP90β-miRNA that suppress HSP90β expression, and cells transfected with HSP90β1-620 that disrupts the interaction of endogenous HSP90β and rapsyn. These results support a working model where agrin stimulates the interaction between HSP90β and rapsyn and thus stabilizes rapsyn. When HSP90β is inhibited or its expression suppressed, rapsyn becomes unstable and degraded possibly via the 26S proteasome system. HSP90β was concentrated at the NMJ (Figures 1F and 1G) and present in agrin-induced AChR clusters (Figure 3B). However, little HSP90β was detectable in spontaneous AChR clusters (Figure S1B), although its association with surface AChR and rapsyn could be demonstrated in naive muscle cells in pull-down and coimmunoprecipitation assays (Figures 1C and 2A). These results could suggest that the amount of HSP90β associated with spontaneous AChR clusters was at low levels that could not be revealed by immunostaining. Nevertheless, these results suggest that HSP90β regulates agrin-regulated AChR clusters. This notion is supported by increased association of HSP90β with clustered AChR (Figure 1C, 1E, and 3B). HSP90β is a molecular chaperone that aids in the folding, assembly-disassembly, and activation of a wide range of substrate or client proteins (Pearl and Prodromou, 2006). Many proteins are known to be enriched at the NMJ, including MuSK (DeChiara et al., 1996, Sanes and Lichtman, 2001), and in particular, several have been identified to associate with the AChR of adult rabbit muscle cells or in cultured muscle cells, including APC, actin, and α-actinin (Mitsui et al., 2000, Wang et al., 2003) (data not shown). We show that HSP90β is recruited to the AChR complex upon agrin stimulation via direct interaction with rapsyn. Inhibition of HSP90β function by 17-AAG or by a dominant-negative mutant or suppression of HSP90β expression by miRNA reduces static levels of rapsyn and its half-life in myotubes. Moreover, rapsyn levels were reduced in muscles that expressed HSP90β-miRNA or the dominant-negative mutant or in muscles from mice that were injected with 17-AAG. These results suggest that HSP90β stabilizes rapsyn and subsequently AChR clustering. Intriguingly, pretreatment of myotubes with 17-AAG also inhibited agrin-induced formation of AChR clusters. This is probably because that the inhibition of HSP90β by 17-AAG is long-lasting. A recent study indicates that 17-AAG accumulates in cells and its intracellular concentration remains high even 72 hr after treatment (Chiosis et al., 2003). Interestingly, agrin stimulation did not have consistent effect on total levels of rapsyn in muscle cells, which could suggest that HSP90β alone is not sufficient for rapsyn folding or stabilization. In support of this notion was that HSP90β overexpression had no consistent effect on levels of rapsyn or its half-life (Figure S12). These observations suggest the existence of additional mechanisms for rapsyn stabilization. In addition to HSP90β, rapsyn also associates with HSP70, another component of the HSP complex (Figures 2A and 2C). HSP70 is known to participate to regulate protein folding and degradation (Bukau and Horwich, 1998, Hartl and Hayer-Hartl, 2002). It is likely that HSP70 participates in rapsyn folding and stabilization.