title: Ribozyme-mediated suppression of the G protein gamma7 subunit suggests a role in hormone regulation of adenylylcyclase activity.
abstract: Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein gamma subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual gamma subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the gamma7 subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted gamma7 subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the gamma7 RNA transcript. In particular, this ribozyme did not result in cleavage of the gamma12 RNA transcript, which is 75% identical to the gamma7 RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the gamma7 subunit in HEK 293 cells. Coincident with this loss in gamma7 subunit, there was a specific reduction in the protein expression of the beta1 subunit, suggesting that the beta1 and gamma7 subunits may functionally interact to form a betagamma dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the gamma7 subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E1-, stimulated adenylylcyclase activity. Suppression of the gamma7 subunit expression had no effect on carbachol- and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual gamma subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the gamma7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.
authors.affiliation: Henry Hood MD Research Program, Pennsylvania State University, College of Medicine, Danville, Pennsylvania 17822, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Mullah
authors.forename: C
authors.initials: C
authors.lastname: Hansen
authors.forename: J
authors.initials: J
authors.lastname: Asundi
authors.forename: J D
authors.initials: JD
authors.lastname: Robishaw
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002217:Carbachol / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D005240:Feasibility Studies; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000318:Adrenergic beta-Agonists; D006728:Hormones; D010716:Phosphatidylinositols; D016337:RNA, Catalytic; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000527:Alprostadil; D007545:Isoproterenol