Abstract
The effect of Gi/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed alpha2A-adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin-sensitive manner. Activation of Gs proteins switched the response of the alpha2A-adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbetagamma scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (alphao1 + beta1gamma2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (alphai1 + beta1gamma2) resulted in a potentiation of both the TPA- and the Gs-stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein-linked signalling.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenylyl Cyclases / metabolism*
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Animals
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Binding, Competitive / drug effects
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Binding, Competitive / physiology
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Cell Line
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Colforsin / pharmacology
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Cyclic AMP / metabolism
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Enzyme Activation / drug effects
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Enzyme Activation / physiology
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Enzyme Activators / pharmacology
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Enzyme Inhibitors / pharmacology
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GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
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GTP-Binding Protein alpha Subunits, Gs / metabolism
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Guanosine 5'-O-(3-Thiotriphosphate) / pharmacokinetics
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Heterotrimeric GTP-Binding Proteins / metabolism*
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Humans
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Idazoxan / analogs & derivatives
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Idazoxan / pharmacokinetics
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Pertussis Toxin / pharmacology
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Protein Kinase C / antagonists & inhibitors
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Receptors, Adrenergic, alpha-2 / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Spodoptera
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Tetradecanoylphorbol Acetate / pharmacology
Substances
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ADRA2A protein, human
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Enzyme Activators
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Enzyme Inhibitors
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Receptors, Adrenergic, alpha-2
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Colforsin
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Guanosine 5'-O-(3-Thiotriphosphate)
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Cyclic AMP
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2-methoxyidazoxan
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Pertussis Toxin
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Protein Kinase C
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GTP-Binding Protein alpha Subunits, Gi-Go
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GTP-Binding Protein alpha Subunits, Gs
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Heterotrimeric GTP-Binding Proteins
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Adenylyl Cyclases
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adenylyl cyclase 2
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Tetradecanoylphorbol Acetate
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Idazoxan