Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein α-subunit (Gαs), G-protein β1γ2 subunit dimer (Gβ1γ2), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Gαs, Gβ1γ2, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Gαs-coupled receptors together with Gαs, Gβ1γ2, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Gαs and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.