The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1/2 phosphorylation

Muscarinic M3 receptors stimulate ERK1/2, the mitogen- activated protein kinase pathway. A mutant of the muscarinic M3 receptor in which most of the third intracellular (i3) loop had been deleted (M3-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells,M3-short greatly reduced the ability ofM3 to activate ERK1/2. In another set of experiments we tested the ability of a mutant M3/M2(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M3 receptor were replaced with the corresponding segment of the muscarinic M2 receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Gq, but it is weakly coupled to Gi. Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M3-short greatly reduced the ability of M3/ M2(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M3 cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 M and a second increase starting from 10 M. In CHO M3-short and in double-transfected CHO M3/M3-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mM carbachol. This suggests that in double-transfected CHO cells M3-short prevents the effect of the higher doses of carbachol on the M3 receptor. In a final experiment we tested the ability of co-transfected chimeric 2/M3 and M3/2 receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a receptor dimer.