title: Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.
abstract: 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 alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), 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, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) 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.
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan. sakihama@med.rcast.u-tokyo.ac.jp
authors.forename: Toshiko
authors.initials: T
authors.lastname: Sakihama
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Masuda
authors.forename: Takato
authors.initials: T
authors.lastname: Sato
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics*; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000235:genetics*; D019151:Peptide Library; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D019151:Peptide Library; D043562:Receptors, G-Protein-Coupled; D000262:Adenylyl Cyclases