imprint:
National Academy of Sciences of the United States of America, 1996
Published in:Proceedings of the National Academy of Sciences of the United States of America
Language:
English
ISSN:
0027-8424
Origination:
Footnote:
Description:
<p>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 ([Ca<sup>2+</sup>]<sub>i</sub>) was measured. Antisense oligonucleotides directed against the mRNA coding for α<sub>q</sub>and α<sub>11</sub>subunits both suppressed the carbachol-induced increase in [Ca<sup>2+</sup>]<sub>i</sub>. In cells injected with antisense oligonucleotides directed against α<sub>o1</sub>and α<sub>14</sub>subunits, the carbachol effect was unchanged. A corresponding reduction of Gα<sub>q</sub>and Gα<sub>11</sub>proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of α<sub>q</sub>and α<sub>11</sub>antisense oligonucleotides. Expression of Gα<sub>q</sub>and Gα<sub>11</sub>completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for β<sub>1</sub>, β<sub>4</sub>, and γ<sub>4</sub>subunits showed a suppression of the carbachol-induced increase in [Ca<sup>2+</sup>]<sub>i</sub>compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the β<sub>2</sub>, β<sub>3</sub>, γ<sub>1</sub>, γ<sub>2</sub>, γ<sub>3</sub>, γ<sub>5</sub>, and γ<sub>7</sub>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 α<sub>q</sub>, α<sub>11</sub>, β<sub>1</sub>, β<sub>4</sub>, and γ<sub>4</sub>to activate phospholipase C.</p>