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Biochemistry. 1999 Feb 23;38(8):2272-8.

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.

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  • 1Receptor Systems Unit, Medicines Research Centre, Glaxo Wellcome Research and Development, Stevenage, England, U.K.

Abstract

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.

PMID:
10029519
DOI:
10.1021/bi982054f
[PubMed - indexed for MEDLINE]
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