Gi alpha 2 mediates the inhibitory regulation of adenylylcyclase in vivo: analysis in transgenic mice with Gi alpha 2 suppressed by inducible antisense RNA.
The role of the GTP-binding regulatory protein (G-protein) Gi alpha 2 in vivo was explored using transgenic mice in which the alpha-subunit of Gi alpha 2 was suppressed by antisense RNA. Rat hepatoma FTO-2B cells provide an ideal test system for constructs employing the expression vector pPCK-AS, designed to express antisense RNA at birth under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cells transfected with the expression vector containing a sequence antisense to Gi alpha 2 (pPCK-ASGi alpha 2) displayed expression of RNA antisense to Gi alpha 2 that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to Gi alpha 2 and suppression of the expression of Gi alpha 2, but not Gsa and Gi alpha 3, was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of Gi alpha 2 in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of Gi alpha 2 in white adipocytes of transgenic mice resulted in 3.1-fold elevation of basal cyclic AMP accumulation. Cyclic AMP accumulation in response to stimulation by epinephrine (10 microM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the Gi alpha 2-deficient cells. The inhibitory adenylylcyclase pathway, in sharp contrast, was severely blunted in response to challenge by the inhibitory A1-purinergic agonist, (-)R-N6-phenylisopropyladenosine. These studies illuminate a critical role of Gi alpha 2 in the inhibitory adenylylcyclase signaling pathway in vivo.
Moxham
C M
CM
Department of Molecular Pharmacology, State University of New York at Stony Brook 11794-8651.
Hod
Y
Y
Malbon
C C
CC
D000262:Adenylyl Cyclases / Q000378:metabolism*; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D004790:Enzyme Induction / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006130:Growth Disorders / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis; D016372:RNA, Antisense / Q000494:pharmacology*; D051381:Rats; D015290:Second Messenger Systems / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured
D016372:RNA, Antisense; C039919:RNA, recombinant; D012313:RNA; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D000262:Adenylyl Cyclases