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Comparative Study
. 2007 Apr 11;27(15):3968-73.
doi: 10.1523/JNEUROSCI.4691-06.2007.

Beta-catenin regulates acetylcholine receptor clustering in muscle cells through interaction with rapsyn

Bin Zhang  1 , Shiwen Luo, Xian-Ping Dong, Xian Zhang, Chunming Liu, Zhenge Luo, Wen-Cheng Xiong, Lin Mei
Affiliations
Comparative Study

Beta-catenin regulates acetylcholine receptor clustering in muscle cells through interaction with rapsyn

Bin Zhang et al. J Neurosci. .

Abstract

Agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor (AChR) clustering at the neuromuscular junction. However, exactly how agrin mediates this effect remains unclear. Here we demonstrate that the beta-catenin interacts with rapsyn, a molecule key for AChR clustering. Agrin stimulation increases the association of beta-catenin with surface AChRs. Suppression of beta-catenin expression inhibited agrin-induced AChR clustering, suggesting a necessary role of beta-catenin in this event. The beta-catenin action did not appear to require the function of T-cell factors (TCFs), suggesting a mechanism independent of TCF-mediated transcription. In contrast, prevention of beta-catenin from interacting with alpha-catenin attenuated agrin-induced AChR clustering. These results suggest that beta-catenin may serve as a link between AChRs and alpha-catenin-associated cytoskeleton, revealing a novel function of beta-catenin in synaptogenesis.

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Figures

Figure 1.
Figure 1.
β-Catenin associates with the AChR complex through rapsyn. A, Direct interaction of β-catenin with rapsyn. [35S]-labeled β-catenin proteins were incubated with GST alone or GST–rapsyn immobilized on beads. Bound [35S]-β-catenin proteins were visualized by autoradiogram. Molecular weight markers are in kilodaltons. B, Schematic diagram of β-catenin constructs and binding activity to rapsyn. C, Association of β-catenin with surface AChR in muscle cells. Live myotubes, treated with agrin for indicated times, were incubated with biotin-αBTX to label surface AChR. Lysates were incubated with streptavidin-coupled agarose beads, and bead-associated proteins were subjected to immunoblotting with antibodies against AChRα, β-catenin, and rapsyn. Lysates (5% of input) were blotted to indicate equal amounts of inputs. D, β-Catenin association with AChR requires rapsyn. Control or rapsyn mutant (R−/−) myotubes were stimulated without or with agrin for 12 h. Proteins associated with surface AChRs were isolated as in C and probed with indicated antibodies. Lysates (5% of input) were blotted to indicate equal amounts of inputs. PD, Pull down; IB, immunoblotting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 2.
Figure 2.
β-Catenin is required for agrin-induced AChR clusters. A, B, β-Catenin shRNAs suppress expression of β-catenin but not rapsyn. COS7 cells were cotransfected with stable GFP–β-catenin (GFP-β-cat*) (A) or GFP-rapsyn (B) with β-catenin shRNA-811, -1196, -2405, or control shRNA-scramble. Lysates of transfectants were subjected to immunoblotting using anti-GFP antibody. C, Refractory GFP-RF811 and GFP-RF1196 were resistant to respective β-catenin shRNA constructs. COS7 cells were cotransfected with GFP-β-cat* or refractory constructs with shRNA-811 or -1196. Lysates of transfectants were subjected to immunoblotting using antibodies against GFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D, Agrin-induced AChR clustering was inhibited by β-catenin shRNAs. C2C12 myoblasts were transfected with pEGFP alone or together with individual shRNA constructs (pEGFP:shRNA, 1:20). Transfected myotubes, labeled by GFP, were stimulated without or with agrin and scored for AChR clusters. Images of representative experiments that were repeated five times with similar results are shown. Histograms (right) show quantification of AChR clusters in each transfection. Open bars, Without agrin; filled bars, agrin stimulated. E, Rescue of β-catenin shRNA-mediated inhibition by refractory constructs. C2C12 myoblasts were transfected with pEGFP together with indicated shRNA and β-catenin expression constructs (pEGFP:β-catenin:shRNA, 1:10:10). AChR clusters were assayed as in D. Histograms (bottom) show quantification of AChR clusters in each transfection. F, β-Catenin shRNA effect on AChR expression. C2C12 myoblasts were transfected by nucleofection with pEGFP alone or together with shRNA constructs. Eight hours after transfection, cells were switched to the DM. Myotube lysates were subjected to immunoblotting using antibodies against β-catenin and AChRα. Band density was analyzed by NIH Image. Data shown in the histograms are mean ± SEM with control as 100% (n = 3). G, Comparison of β-catenin shRNA effects on AChR levels and clusters. Data on AChR levels are from F, whereas data on AChR clusters are from D. All quantitative data are shown as mean ± SEM. *p < 0.01. IB, Immunoblotting.
Figure 3.
Figure 3.
β-Catenin transcription activity is dispensable in regulation of AChR clustering. A, Inhibition of TopFlash reporter activity by dn-TCF4. COS7 cells were transfected with TopFlash with or without β-catenin and wild-type (wt)- or dn-TCF. pRL-TK was cotransfected as control of transfection efficiency and sample handling. The ratio was 10:10:1 for TopFlash:TCF contructs:pRL-TK. Relative luciferase activities (firefly/Renila; mean ± SEM) from a representative experiment in duplicates, which was repeated three times with similar results, are shown. B, Normal agrin-induced AChR clusters in C2C12 cells expressing dn-TCF4. C2C12 myoblasts were transfected with pEGFP, dn-TCF4, or wt-TCF4 in a ratio of 1:20 (pEGFP:TCF4 constructs). AChR clusters were assayed as in Figure 2 D. C, Quantitative analysis of data in B. Data were shown as mean ± SEM (n = 30). Open bars, Without agrin; filled bars, agrin stimulated. D, Expression of Wnt, DKK, and Fz-Fc constructs in C2C12 cells. C2C12 myoblasts were transfected with pFLAG-DKK1, hIgG-mFz8CRD, pKH3-Wnt1, pKH3-Wnt4, pKH3-Wnt6, or pKH3-Wnt7b. Expression in resulting myotubes was analyzed by immunoblotting with indicated antibodies. E, Normal AChR clustering by agrin in C2C12 cells expressing Wnt signaling molecules. C2C12 myoblasts were transfected with pEGFP alone or together with indicated constructs in a ration of 1:20. AChR clusters were assayed as in Figure 2 D. F, Quantitative analysis of data in B. Data were shown as mean ± SEM (n = 30). Open bars, Without agrin; filled bars, agrin stimulated. IB, Immunoblotting.
Figure 4.
Figure 4.
β-Catenin regulation of AChR clustering requires interaction with α-catenin. A, Association of α-catenin with surface AChR complex in muscle cells. Surface AChR was labeled and purified from control C2C12 myotubes (left) or those differentiated from cells nucleofected with GFP–β-catenin-AA (GFP-β-Cat-AA; right), as described in Figure 1 C. AChR-associated proteins were analyzed by immunoblotting with antibodies against AChRα and α-catenin. Lysates (5% of input) were also subjected to immunoblotting to indicate equal amounts of inputs. B, Expression of β-catenin-AA inhibits AChR clustering. C2C12 myoblasts were transfected with GFP–β-cat* or GFP–β-Cat-AA. AChR clusters were induced and scored as in Figure 2 D. Images of representative experiments that were repeated five times with similar results, are shown. Histograms show quantification of AChR clusters (mean ± SEM; n = 30; *p < 0.01). C, Working model. Via interacting with rapsyn and α-catenin, β-catenin may link the AChR to the cytoskeleton. β-Catenin may also regulate expression of synaptic proteins including the AChR. PD, Pull down; IB, immunoblotting.
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