Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/oα G proteins following coexpression of receptor and c protein and expression of A1 adenosine receptor-Gi/oα fusion proteins

HEK293T cells were transiently transfected to express either the human A(1) adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the a subunits of G(i1) (C351G), G(i2) (C352G), and G(i3) (C351G) and wild-type G(o1)alpha or fusion proteins comprising the A(1) adenosine receptor and these G(i/o) G proteins to compare A(1) 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 [S-35]-GTP gamma S binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A(1) adenosine receptor and G(i/o) G proteins either as fusion proteins or as separate polypeptides, However, the magnitude of agonist-mediated activation of GTP gamma S binding was greatly enhanced by expressing the A(1) 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. Na preferential activation of any G protein was observed with more than 40 A(1) 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 A(1) adenosine receptor couples equally well to the G(i/o) G proteins G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(o1)alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A(1) adenosine receptor-G(i/o) G protein combination.