A Heterotrimeric G Protein Complex Couples the Muscarinic m1 Receptor to Phospholipase C-β

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-β by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for αqand α11subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against αo1and α14subunits, the carbachol effect was unchanged. A corresponding reduction of Gαqand Gα11proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of αqand α11antisense oligonucleotides. Expression of Gαqand Gα11completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for β1, β4, and γ4subunits showed a suppression of the carbachol-induced increase in [Ca2+]icompared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the β2, β3, γ1, γ2, γ3, γ5, and γ7subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits αq, α11, β1, β4, and γ4to activate phospholipase C.