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 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 alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 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 beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 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), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.
Dippel
E
E
Institut für Pharmakologie, Freie Universität Berlin, Federal Republic of Germany.
Kalkbrenner
F
F
Wittig
B
B
Schultz
G
G
D000818:Animals; D001483:Base Sequence; D002217:Carbachol / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018727:Muscarinic Antagonists / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D054799:Phospholipase C beta; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D018727:Muscarinic Antagonists; D016376:Oligonucleotides, Antisense; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D002217:Carbachol; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; C518036:PLCB2 protein, human; C518039:PLCB3 protein, human; C518042:PLCB4 protein, human; D054799:Phospholipase C beta; C518035:Plcb1 protein, rat; C518038:Plcb2 protein, rat; C518041:Plcb3 protein, rat; C518044:Plcb4 protein, rat; D019204:GTP-Binding Proteins