Abstract

Allosteric models of G protein-coupled receptors predict that G protein influences 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 content should influence the binding properties of these ligands. To test this hypothesis, we systematically assessed the effect of G proteins on the binding of β₂-adrenergic ligands with distinct levels of inverse efficacy. Recombinant baculoviruses encoding the human β₂-adrenoreceptor (β₂AR) were expressed alone or in combination with G protein subunits in Sf9 cells. Coexpression with the G protein αsβ1γ2 did not influence the relative efficacy of the ligands to inhibit the adenylyl cyclase but induced considerable decrease in number of sites detected by [³H]ICI 118551, [³H]propranolol, and¹²⁵I-cyanopindolol. This loss was proportional to the inverse efficacy of the ligand used as the radiotracer in the assay. The addition of Gpp(NH)p inhibited the effects of G protein overexpression indicating that the G proteins acted allosterically. Consistent with this notion, Western blot analysis revealed that coexpression with the G proteins was not accompanied by a loss of immunoreactive β₂AR. Such allosteric effects of the G proteins were also observed in mammalian cells expressing endogenous level of G proteins indicating that the phenomenon is not unique 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.