It is worth pointing out that
It is worth pointing out that phenotypes of in vivo HSP90β inhibition were not identical to those observed in rapsyn mutant mice (Gautam et al., 1995). Postsynaptically, junctional AChR clusters appeared fragmented, in addition to expected reduction in AChR intensity, in muscles injected with 17-AAG or expressing the dominant-negative mutant or HSP90β-miRNA (Figures 4C, 6D, and 5E). Furthermore, in vitro studies showed that some AChR clusters disappeared whereas others reduced in intensity in myotubes (Figure S6 time lapse). This binary effect and fragmentation of AChR clusters could suggest a regulatory role of HSP90β in the stabilization of clusters or the NMJ, the underlying mechanisms of which, however, warrant further investigation. It is possible that HSP90β may regulate the function or stability of other proteins in addition to rapsyn. Candidates on this list include α-dystrobrevin and α-syntrophin, which have been shown to regulate the stabilization of AChR clusters (Adams et al., 2000, Banks et al., 2003, Grady et al., 2000, Pawlikowski and Maimone, 2008, Sadasivam et al., 2005). In rapsyn mutant mice, motor someone’s name grow excessively over the entire muscle with little presynaptic differentiation (Gautam et al., 1995). However, nerve branches in 17-AAG-treated animals appeared to be similar to those in control mice (Figure 4A and 4A′). The lack of presynaptic phenotypes may be due to the time of 17-AAG injection, i.e., E14.5, being after the formation of primitive AChR clusters (Lin et al., 2001, Yang et al., 2001) and incomplete ablation of AChR clusters. It is also possible that HSP90β inhibition reduces axon mobility or suppresses expression of axon attractive molecules or trophic factors in muscles. HSP90β inhibition did not appear to alter the levels of MuSK that is enriched in the postsynaptic membrane (Figures 3C and 3E). No consistent effect of HSP90β inhibition was observed on expression of signaling molecules that have been implicated in AChR clustering, including Abl, Rac, and Cdc42 (Figure 3D) or myosin heavy chain (MHC), MyoD, or myogenin (Figure S7A). These observations indicate the specificity of HSP90β-dependent protein stability, suggesting the necessity of direct interaction for HSP90β regulation. Rapsyn has been shown to confer AChR stability (Banks et al., 2003, Willmann and Fuhrer, 2002). However, AChR is much more stable than rapsyn, with a half-life of ∼24 hr in muscle cells (Berg and Hall, 1975, Devreotes and Fambrough, 1975, Gervasio and Phillips, 2005, Wang et al., 1999). The receptor becomes more stable when clustered or at the NMJ, with a half-life of ∼10 days (Levitt et al., 1980, Salpeter and Loring, 1985). Therefore, we anticipate reduced levels of AChR after long-term inhibition of HSP90β. HSP90 has three regions: N-terminal nucleotide binding pocket for ATP and geldanamycin, the middle region for substrate/client proteins, and the C-terminal region that interact with cochaperone proteins such as HSP70, HSP40, and p23 (Pearl and Prodromou, 2006). The interaction with the cochaperones has been shown to regulate proper folding or function of substrate proteins. In line with this notion, we found that the C-terminal truncation mutant of HSP90β1-620 was unable to maintain rapsyn stability, although it was able to bind with rapsyn (Figures 2E). In fact, due to its ability to interact with rapsyn, expressed HSP90β1-620 attenuates the interaction of endogenous rapsyn and HSP90β and thus inhibits agrin-induced AChR cluster formation. These results demonstrate that the C terminus of HSP90β is involved in regulating the stability of rapsyn, suggesting the possible involvement of other chaperone proteins. Note that HSP90β overexpression did not appear to alter agrin-induced AChR clusters (Figure 6B) and had no consistent effect on levels of rapsyn or its half-life (Figure S12), suggesting that the limiting factor may not be the levels of HSP90β, but the regulated interaction with rapsyn. Exactly how rapsyn is degraded after dissociation from HSP90β remains to be fully elucidated. The inhibition of 17-AAG-induced depletion of rapsyn by MG-132 suggests that degradation occurs via 26S proteasomal-dependent hydrolysis. This conclusion is further supported by our observations that the lysosomal protease inhibitors, chloroquine and monensin, had no effect on 17-AAG-induced loss of rapsyn.