title: The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1/2 phosphorylation.
abstract: Muscarinic M(3) receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the muscarinic M(3) receptor in which most of the third intracellular (i3) loop had been deleted (M(3)-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, M(3)-short greatly reduced the ability of M(3) to activate ERK1/2. In another set of experiments we tested the ability of a mutant M(3)/M(2)(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M(3) receptor were replaced with the corresponding segment of the muscarinic M(2) receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Galpha(q), but it is weakly coupled to Galpha(i). Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M(3)-short greatly reduced the ability of M(3)/M(2)(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M(3) cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 microm and a second increase starting from 10 microm. In CHO M(3)-short and in double-transfected CHO M(3)/M(3)-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 M(3)-short prevents the effect of the higher doses of carbachol on the M(3) receptor. In a final experiment we tested the ability of co-transfected chimeric alpha(2)/M(3) and M(3)/alpha(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.
authors.affiliation: Department of Neurosciences, University of Pisa, 56100 Pisa, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Novi
authors.forename: Marco
authors.initials: M
authors.lastname: Scarselli
authors.forename: Giovanni U
authors.initials: GU
authors.lastname: Corsini
authors.forename: Roberto
authors.initials: R
authors.lastname: Maggio
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D019281:Dimerization; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007163:Immunosorbent Techniques; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D011993:Recombinant Fusion Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases