FIG. 9.

The agrin-triggered autonomous pathway is maximally activated if MuSK phosphorylation reaches a certain level. (A) C2 myotubes were continuously treated for 5 min, 40 min, or overnight with 0.1 or 0.5 nM agrin, and cell lysates were analyzed by MuSK immunoprecipitation (IP) and phosphotyrosine immunoblotting (pTyr-blot). As control, MuSK antibody was omitted (−Ab). (B) C2 myotubes were treated for 5 or 40 min with 0.1 or 0.5 nM agrin, followed by withdrawal for 8 h. AChRs were stained with rhodamine-α-BT and were analyzed by fluorescence microscopy. Within each pulse, the numbers of AChR clusters per myotube treated with 0.5 nM agrin, quantitated as described for Fig. 5, were set to 100%. *P < 0.02 by two-tailed paired t test. (C) Myotubes were treated for 5 min with 0.1 nM (diamonds) or 0.5 nM (squares) agrin, followed by 10 min, 35 min, or 8 h of agrin withdrawal, MuSK immunoprecipitation, and phosphotyrosine immunoblotting. Parallel cells were continuously treated for 40 min with 0.5 nM agrin (triangle). After a 5-min agrin pulse, MuSK phosphorylation increased in the withdrawal period. Quantitation shows the means ± SEM of three experiments evaluated by densitometric scanning. The area between the drawn lines indicates a critical level of MuSK phosphorylation. Above this level, maximal clustering occurs even in the subsequent absence of agrin.