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

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 G_i1 (C351G), G_i2 (C352G), and G_i3 (C351G) and wild-type G_o1α or fusion proteins comprising the A₁ adenosine receptor and these G_i/o G proteins to compare A₁ adenosine receptor agonist-mediated activation of these G_i family G proteins upon coexpression of individual G_i/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 G_i/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 G_i 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 G_i/o G proteins G_i1α, G _i2α, G_i3α, and G_o1α, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A₁ adenosine receptor−G_i/o G protein combination.