Comparative Analysis of the Efficacy of A1 Adenosine Receptor Activation of Gi/oα G Proteins following Coexpression of Receptor and G Protein and Expression of A1 Adenosine Receptor−Gi/oα Fusion Proteins

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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 α subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1α 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]GTPγS 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 GTPγS 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 Gi1α, G i2α, Gi3α, and Go1α, 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.