Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/o alpha G proteins following coexpression of receptor and G protein and expression of A1 adenosine receptor-Gi/o alpha fusion proteins.
HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the alpha subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1alpha or fusion proteins comprising the A1 adenosine receptor and these Gi/o G proteins to compare A1 adenosine receptor agonist-mediated activation of these Gi family G proteins upon coexpression of individual Gi/o G proteins and receptor versus expression as receptor-G protein fusion proteins. Addition of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) to membranes of pertussis toxin-treated cells resulted in a concentration-dependent stimulation of [35S]GTPgammaS binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A1 adenosine receptor and Gi/o G proteins either as fusion proteins or as separate polypeptides. However, the magnitude of agonist-mediated activation of GTPgammaS binding was greatly enhanced by expressing the A1 adenosine receptor and Gi family G proteins from chimaeric open reading frames. This observation was consistent following the study of more than 40 agonists. No preferential activation of any G protein was observed with more than 40 A1 receptor agonists following cotransfection of receptor with G protein or transfection of receptor-G protein fusion proteins. These studies demonstrate the utility of using fusion proteins to study receptor-G protein interaction, show that the A1 adenosine receptor couples equally well to the Gi/o G proteins Gi1alpha, G i2alpha, Gi3alpha, and Go1alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A1 adenosine receptor-Gi/o G protein combination.
Receptor Systems Unit, Medicines Research Centre, Glaxo Wellcome Research and Development, Stevenage, England, U.K.
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Wise
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Sheehan
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Rees
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Lee
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Milligan
D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D004622:Embryo, Mammalian; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D058906:Purinergic P1 Receptor Agonists; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D019830:Adenosine-5'-(N-ethylcarboxamide); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go