title: Functional reconstitution of olfactory receptor complex on baculovirus.
abstract: Despite that recent progress in genomics has elucidated the genomic structure of the olfactory receptors (ORs), most of them are still orphan receptors. The low expression level of ORs in heterologous cells has hampered many attempts to establish cell biological OR assay systems. Recently, we demonstrated that certain G protein-coupled receptors, such as the leukotriene B4 receptor or the dopamine D1 receptor, were efficiently reconstituted on baculovirus budding from infected Sf9 cells. The budded virus (BV) was shown to be mostly free of exogenous proteins other than those related to viral infection, resulting in low-noise assay conditions. Taking advantage of these conditions, we attempted to reconstitute OR complexes on BV. Sf9 cells were coinfected with recombinant baculoviruses harboring the cDNAs encoding adenylyl cyclase, trimeric G-protein, and the receptor: mOR-EG or S6. The coexpression of these proteins was detected by western blot, and the agonist- or antagonist-dependent receptor response was confirmed using ligand-dependent cyclic AMP production. These results demonstrated the successful reconstitution of functional OR complex on BV. Additionally, the expression of OR8B3 on BV, one of human orphan ORs, was also confirmed. This BV expression system is expected to be a highly effective tool for screening unknown ligands for ORs.
authors.affiliation: Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Tokyo 153-8904, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Mitsui
authors.forename: Toshiko
authors.initials: T
authors.lastname: Sakihama
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Takahashi
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Masuda
authors.forename: Rie
authors.initials: R
authors.lastname: Fukuda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hamana
authors.forename: Takaaki
authors.initials: T
authors.lastname: Sato
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D008840:Microfilament Proteins
chemical_list: D000242:Cyclic AMP; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D018035:Receptors, Odorant; D011994:Recombinant Proteins; D019893:Ribosomal Protein S6 Kinases; C544141:Olfr73 protein, mouse; D008840:Microfilament Proteins