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First published online 13 February 2007
doi: 10.1242/jcs.03367

Journal of Cell Science 120, 868-875 (2007)
Published by The Company of Biologists 2007
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Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways

Christi A. Weston1,2,*, Getu Teressa1,2, Benjamin S. Weeks3 and Joav Prives1,{ddagger}

1 Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA
2 Medical Scientist Training Program, Stony Brook University, Stony Brook, NY 11794, USA
3 Department of Biology, Adelphi University, Garden City, NY 11530, USA


Figure 1
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  		Fig. 1. Dominant interfering Rac and Cdc42 block the ability of laminin to cluster AChR. (A) In order to assess the contribution of Rac and Cdc42 activation to laminin-induced AChR clustering, vector (a,b), T7-tagged RacN17 (c,d) or myc-tagged Cdc42N17 (e,f) were transfected into C2 muscle cell cultures. Three days after transfection, the effects of AChR surface distribution in laminin-treated and -untreated myotubes were examined by fluorescence microscopy of cultures surface-labeled with TMR-Bgt. Those myotubes expressing the transfected constructs were identified using indirect immunofluorescence with anti-T7 or anti-myc antibodies, and FITC-labeled secondary antibody (a,c,e). Myotubes expressing RacN17 or Cdc42N17 did not display microclusters (arrowheads) or clusters (arrows) of AChR after laminin treatment (b,d,f); however, adjacent, non-transfected cells were able to cluster AChRs in response to laminin. (B) Quantitative comparison of the number of AChR clusters on the surface of transfected myotubes expressing the Rac and Cdc42 mutants versus vector-transfected myotubes clearly documented the blocking effect of the dominant interfering RacN17 and Cdc42N17 on laminin-induced AChR aggregation (error bars represent ±s.e.m., n=40 cells from five or more separate platings).

 

Figure 2
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  		Fig. 2. Laminin activates Rac and Cdc42 in C2 myotubes. Laminin-induced activation of endogenous Rac (A) and Cdc42 (B) in C2 myotubes was measured using GST-PBD and western blotting with anti-Rac or anti-Cdc42 antibodies. Laminin activates Rac and Cdc42 similarly to agrin-induced Rac and Cdc42 activation in differentiated muscle cells.

 

Figure 3
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  		Fig. 3. Laminin activates JNK in C2 myotubes. (A) JNK activation by laminin was observed in differentiated C2 myotubes but not in undifferentiated myoblasts. (B) The time course of laminin stimulation of c-Jun phosphorylation in myotubes shows a transient response to laminin that results in c-Jun phosphorylation that reaches a maximum within 15 minutes and subsequently declines to baseline levels by 60 minutes.

 

Figure 4
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  		Fig. 4. Laminin activation of JNK is dependent on Rac and Cdc42 activation. (A) C2 cells were transfected with FLAG epitope-tagged JNK alone or in combination with expression plasmids encoding T7-epitope-tagged RacN17 or RacV12. Myotubes were treated for 15 minutes with laminin (10 nM), where specified, and the transfected JNK was immunoprecipitated from cell lysates with anti-FLAG antibody. The immunopurified JNK was incubated with [{gamma}-32P]ATP and GST-c-Jun as a substrate. Levels of transfected Rac and JNK expression were determined by western blotting with anti-T7 and anti-JNK1 primary antibodies, respectively. GST-c-Jun phosphorylation was visualized by autoradiography. As quantitated by a PhosphorImager, cells transfected with JNK alone showed a threefold increase in c-Jun phosphorylation when treated with laminin. By contrast, cells that were cotransfected with JNK plus RacN17 showed no JNK activation when treated with laminin. (B) C2 cells were cotransfected with FLAG epitope-tagged JNK and myc epitope-tagged Cdc42N17 or Cdc42V12. Immunoprecipitated JNK was incubated with [{gamma}-32P]ATP and GST-c-Jun. As quantitated by PhosphorImager, the threefold increase in phosphorylation induced by laminin was eliminated by the dominant negative Cdc42 mutant. Constitutively active mutants of Rac (RacV12) and Cdc42 (Cdc42V12) serve as positive controls for JNK activation. The fold-increases in activity in this figure were consistent over at least five separate experiments.

 

Figure 5
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  		Fig. 5. Dominant interfering Rho inhibits the ability of laminin to cluster AChR. (A) In order to assess the contribution of Rho activation to laminin-induced AChR clustering, differentiated muscle cells were microinjected with RhoN19 or vector. After microinjection, the cells were treated with laminin (10 nM) overnight. One day after injection, the effects of AChR surface distribution in RhoN19-expressing (c,d) versus vector-expressing cells (a,b) treated with laminin were examined by confocal microscopy of cultures surface-labeled with TMR-Bgt. Those myotubes injected with RhoN19 were identified with co-injected FITC-goat anti-mouse antibody (a,c). Myotubes expressing RhoN19 did not display full-sized AChR clusters after treatment with laminin (d), compared with those myotubes injected with vector only (b). Scale bar, 10 µm. (B) Quantitative comparison of the number of AChR clusters on the surface of myotubes expressing the Rho mutant versus control myotubes clearly documents the inhibiting effect of the dominant interfering RhoN19 on AChR cluster (black bars) and microcluster (grey bars) formation in response to laminin (10 nM) treatment (error bars represent ±s.e.m.; n=40 cells from five or more separate platings).

 

Figure 6
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  		Fig. 6. C3 transferase impairs laminin-induced AChR clustering. (A) To confirm the results found with RhoN19 on laminin-induced AChR clustering, C3 transferase was added to C2 myotubes 3 hours prior to overnight laminin (10 nM) treatment. The effects on AChR surface distribution were examined by confocal microscopy of cultures surface-labeled with TMR-Bgt. C3 impairs the ability of agrin and laminin to form AChR clusters (b,d) compared with cells that were not treated with C3 (a,c). (B) Quantitative analysis of the effect of C3 on clustering of AChR clearly shows that inactivation of Rho impairs the formation of AChR clusters in response to laminin (10 nM) and agrin (5 nM) (error bars represent ±s.e.m.; n=100 cells from five or more separate platings).

 

Figure 7
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  		Fig. 7. Laminin stimulates increased binding of Rho to the Rho-binding domain of Rhotekin in C2 myotubes. Laminin-induced Rho activation in nontransfected C2 myotubes was measured by the increase of endogenous Rho bound to GST-TRBD. Cultures were treated with laminin (10 nM) for 15 minutes, and lysates were incubated with GST-TRBD and western blotted with antibody to Rho. Laminin caused increased association of Rho with GST-TRBD in myotubes that is similar to that activation of Rho seen with agrin.

 

Figure 8
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  		Fig. 8. Inhibition of ROCK impairs laminin-induced AChR clustering. To determine the contribution of ROCK to AChR clustering, the ROCK inhibitor Y27632 was added to differentiated C2 muscle cell cultures at a concentration of 20 µM. One day after treatment, the effect on laminin-induced AChR surface distribution in Y27632-treated and -untreated myotubes was quantitated and compared with the effect of ROCK inhibition on agrin-induced clustering. Inhibition of ROCK activation impaired agrin-induced AChR clustering by more than 80% (b), but only impaired laminin-induced AChR clusters by approximately 40% (d) compared with untreated cells (a,c) (error bars represent ±s.e.m.; n=100 cells from five or more separate platings).

 

Figure 9
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  		Fig. 9. Inhibition of laminin-induced complex AChR clustering by RhoN19. (A) To determine whether laminin-induced complex AChR cluster formation is dependent on activation of the Rho GTPases, as are soluble laminin-induced AChR clusters, C2 myotubes transfected with GFP-tagged RhoN19 were replated on laminin-coated coverslips. Cultures were surface-labeled with TMR-Bgt and examined at day 4 post-replating by confocal microscopy. Myotubes expressing RhoN19 (a) did not display AChR clusters (b) compared with nontransfected adjacent cells that formed complex clusters. (B) Quantitative comparison of the number of AChR clusters on the surface of transfected myotubes expressing the Rho mutant (hatched bars) versus control myotubes (black bars) clearly documents the inhibiting effect of the dominant interfering RhoN19 on complex AChR cluster formation in response to substrate laminin at all stages of development (error bars represent ±s.e.m.; n=40 cells from five separate platings).

 

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© The Company of Biologists Ltd 2007