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. 2010 May 12;30(19):6620–6634. doi: 10.1523/JNEUROSCI.5778-09.2010

Figure 7.

Figure 7.

Erbin interacts concomitantly with MuSK and ErbB2. a, Scheme of full-length mouse Erbin and deletion mutants thereof. The C-terminal part of Erbin is encoded by exons 21–26. Below this scheme, mouse or human Erbin mutants lacking different single exons are depicted. Other human Erbin mutants consist only of the MuSK binding domain or the PDZ domain. b, Expression of these different mouse and human Erbin variants, as presented in a, was ascertained by Western blot. c, Precipitation of the human Erbin MuSK binding domain (hErbin-MuSK-bd) using transiently transfected HEK293 cells coprecipitated full-length MuSK as shown by Western blot. d, Extracts from transiently transfected HEK293 cells expressing different Erbin variants were used for coimmunoprecipitation studies. After precipitation of Erbin variants, precipitates were analyzed for the presence of full-length MuSK by Western blot. Note that precipitation of any human Erbin mutant that does not contain the MuSK binding domain is unable to coprecipitate MuSK. e, GST fusions of human Erbin-NΔ-1007, Erbin-PDZ, or Erbin-ΔPDZ, immobilized on glutathione beads, were incubated with HEK293 cell extracts containing full-length ErbB2 and MuSK and bound proteins were analyzed by Western blot. Note that GST-hErbin-PDZ pulled down ErbB2 but not MuSK, while GST-hErbin-ΔPDZ pulled down MuSK but not ErbB2. Further, the specificity of this binding was demonstrated by competition with either human Erbin-PDZ or Erbin-MuSK-bd, which inhibited the pull down of either ErbB2 or MuSK, respectively. f, To investigate whether overexpression of Erbin variants affects AChR gene expression, C2C12 cells were transfected three independent times by a hErbin-v2, hErbin-ΔMuSK-bd, hErbin-MuSK-bd, or hErbin-PDZ together with an AChRε-luciferase reporter. Moreover, the same transfections were done together with an additional plasmid encoding constitutively active ErbB2 (neuT). Luciferase activities were measured from myoblasts (MB) or from myotubes (MT). Note that the hErbin-MuSK-bd inhibited the reporter in myotubes that were cotransfected with neuT, but not in myoblasts.