Arecoline, a muscarinic agonist, activates MPK-1 by increasing its phosphorylation. This activation is significantly reduced in a ras partial loss-of-function mutant, and completely blocked by co-treatment with a MEK inhibitor U0126. These results suggest that acetylcholine signals to MPK-1 through the conventional ras-raf-mek-mapk pathway. Co-treatment with atropine, a muscarinic receptor antagonist can reduce the activation, implying that this activation is through muscarinic receptors. Among the three muscarinic receptors, we think GAR-2 and GAR-3 are involved in MPK-1 signaling. There are two reasons we think GAR-2 is involved. First, GAR-2 is expressed mostly in neurons based on its gfp expression, which is similar to either MPK-1:gfp or antibody staining of a phospho MPK-1 antibody after 1mM arecoline treatment. Second, a
gar-2 deletion mutant shows reduction of
mpk-1 activation after 1mM arecoline treatment. Another piece of indirect evidence is that a TPA-1 mutant shows decreased MPK-1 activation, whereas phorbol ester can activate MPK-1. Since GAR-2, TPA-1 and MPK-1 are predominately expressed in neurons, we hypothesize that GAR-2 activates MPK-1 through TPA-1 in neurons. A
gar-3 deletion mutant doesnt show as significant reduction of MPK-1 activation as
gar-2. However, we think GAR-3 could also be involved in MPK-1 activation by arecoline because of following. Kate Steger has shown that the arecoline hypersensitivity of
gpb-2 can be suppressed by a mutation in
gar-3, as well as by a mutation in Gq (
egl-30).
gpb-2 mutants are hypersensitive to arecoline: they arrest in the L1 stage and eventually die in the presence of 5mM arecoline, due to a pumping defect caused by excessive Gq signaling in the pharyngeal muscle. When we tested MPK-1 activation in the
gpb-2 mutant, the
gpb-2 mutant, as well as a Gq gain of function mutant or a ras gain of function mutant, shows hyperactivation of MPK-1. Introduction of a reduced function mutation of
mpk-1 can rescue arecoline hypersensitivity of the
gpb-2 mutant. In summary, we think that muscarinic receptors can activate MPK-1 both in pharyngeal muscle and neurons through different receptors and intermediates. GAR-3 is coupled to Gq and MPK-1 could be a downstream target in pharyngeal muscle. GAR-2, on the other hand, may couple acetylcholine signaling to MPK-1 in neurons. Our studies suggest that MPK-1 may be able to function differently in response to different receptors in different cells.