Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells

• Published in: Receptors & channels Vol. 10; no. 3-4; p. 117
• Main Authors: Ames, Robert, Nuthulaganti, Parvathi, Fornwald, Jim, Shabon, Usman, van-der-Keyl, Harjeet, Elshourbagy, Nabil
• Format: Journal Article
• Language: English
• Published: England 2004
• Subjects: Humans; Osteosarcoma - genetics; Osteosarcoma - metabolism; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Lysophosphatidic Acid - genetics; Receptors, Lysophosphatidic Acid - metabolism; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Receptors, Neurokinin-1 - genetics; Receptors, Neurokinin-1 - metabolism; Receptors, Neurokinin-2 - genetics; Receptors, Neurokinin-2 - metabolism; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Tumor Cells, Cultured
• ISSN: 1060-6823
• DOI: 10.1080/10606820490515012

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.