Giα2 mediates the inhibitory regulation of adenylylcyclase in vivo: Analysis in transgenic mice with Giα2 suppressed by inducible antisense RNA

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Abstract
The role of the GTP-binding regulatory protein (G-protein) Giα2 in vivo was explored using transgenic mice in which the α-subunit of Giα2 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 Giα2 (pPCK-ASGiα2) displayed expression of RNA antisense to Giα2 that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to Giα2 and suppression of the expression of Giα2, but not Gsa and Giα3, was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of Giα2 in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of Giα2 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 (10μM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the Giα2-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 Giα2 in the inhibitory adenylylcyclase signaling pathway in vivo. © 1993Wiley-Liss, Inc.