title: Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells.
abstract: Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.
authors.affiliation: Gene Expression & Protein Biochemistry, Discovery Research Biology, GlaxoSmithKline, King of Prussia, Pennsylvania, USA. bob.ames@gsk.com
authors.forename: Robert
authors.initials: R
authors.lastname: Ames
authors.forename: Parvathi
authors.initials: P
authors.lastname: Nuthulaganti
authors.forename: Jim
authors.initials: J
authors.lastname: Fornwald
authors.forename: Usman
authors.initials: U
authors.lastname: Shabon
authors.forename: Harjeet
authors.initials: H
authors.lastname: van-der-Keyl
authors.forename: Nabil
authors.initials: N
authors.lastname: Elshourbagy
mesh_terms: D006801:Humans; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D049368:Receptors, Lysophosphatidic Acid / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D018041:Receptors, Neurokinin-2 / Q000235:genetics / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011976:Receptors, Muscarinic; D018040:Receptors, Neurokinin-1; D018041:Receptors, Neurokinin-2; D018048:Receptors, Purinergic P2