A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic mi receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic mi 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 alpha(q) and alpha(11) subunits both suppressed the carbachol-induced increase in [Ca2+](i). In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha(14) subunits, the carbachol effect was unchanged. A corresponding reduction of G alpha(q) and G alpha(11) proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha(11) antisense oligonucleotides. Expression of G alpha(q) and G alpha(11) completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta(1), beta(4), and gamma(4) subunits showed a suppression of the carbachol-induced increase in [Ca2+](i) compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta(2), beta(3), gamma(1), gamma(2), gamma(3), gamma(5), and gamma(7) subunits, 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 alpha(q), alpha(11), beta(1), beta(4), and gamma(4) to activate phospholipase C.