Allosteric effects of G protein overexpression on the binding of beta-adrenergic ligands with distinct inverse efficacies

Allosteric models of G protein-coupled receptors predict that G protein inf luences the spontaneous isomerization between inactive (R) and active (R-*) conformations. Since inverse agonists have been proposed to preferentially bind to the inactive and uncoupled form(s), changes in the G protein conte nt should influence the binding properties of these ligands. To test this h ypothesis, we systematically assessed the effect of G, proteins on the bind ing of beta (2)-adrenergic ligands with distinct levels of inverse efficacy . Recombinant baculoviruses encoding the human beta (2)-adrenoreceptor (bet a (2)AR) were expressed alone or in combination with G protein subunits in Sf9 cells. Coexpression with the G protein alphas beta1 gamma2 did not infl uence the relative efficacy of the ligands to inhibit the adenylyl cyclase but induced considerable decrease in number of sites detected by [H-3]ICI 1 18551, [H-3]propranolol, and I-125-cyanopindolol. This loss was proportiona l to the inverse efficacy of the ligand used as the radiotracer in the assa y. The addition of Gpp(NH)p inhibited the effects of G protein overexpressi on indicating that the G proteins acted allosterically. Consistent with thi s notion, Western blot analysis revealed that coexpression with the G prote ins was not accompanied by a loss of immunoreactive beta (2)AR. Such allost eric effects of the G proteins were also observed in mammalian cells expres sing endogenous level of G proteins indicating that the phenomenon is not u nique to overexpression systems. Taken together, these results demonstrate that the apparent receptor number detected by radiolabeled inverse agonists is affected by the content in G proteins as a result of their influence on R/R-* isomerization.