A HETEROTRIMERIC G-PROTEIN COMPLEX COUPLES THE MUSCARINIC M1 RECEPTORTO PHOSPHOLIPASE C-BETA

We addressed the question as to which subtypes of G protein subunits m ediate the activation of phospholipase C-beta by the muscarinic mi rec eptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stabl y transfected with the human muscarinic mi receptor cDNA. We microinje cted antisense oligonucleotides into the nuclei of the cells to inhibi t selectively the expression of G protein subunits; 48 hr later muscar inic receptors were activated by carbachol, and the increase in free c ytosolic calcium concentration ([Ca2+](i)) was measured. Antisense oli gonucleotides 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 7 0-80% compared to uninjected cells was immunochemically detected 2 day s after injection of a mixture of alpha(q) and alpha(11) antisense oli gonucleotides. Expression of G alpha(q) and G alpha(11) completely rec overed after 4 days. Cells injected with antisense oligonucleotides di rected against the mRNAs encoding for beta(1), beta(4), and gamma(4) s ubunits 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 oligonucleoti des 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 sub units alpha(q), alpha(11), beta(1), beta(4), and gamma(4) to activate phospholipase C.