title: A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines.
abstract: Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.
authors.affiliation: Department of Zoology, University of British Columbia, 6270 University Boulevard, Rm 3444, Vancouver, British Columbia, Canada, V6T 1Z4.
authors.forename: Peter J K
authors.initials: PJ
authors.lastname: Knight
authors.forename: Tom A
authors.initials: TA
authors.lastname: Pfeifer
authors.forename: Tom A
authors.initials: TA
authors.lastname: Grigliatti
mesh_terms: D000331:Aequorin / Q000032:analysis; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D002461:Cell Line, Transformed; D018491:Dopamine Agonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000032:analysis; D020962:Heterotrimeric GTP-Binding Proteins / Q000032:analysis / Q000502:physiology; D006801:Humans; D007093:Imidazoles; D008024:Ligands; D009036:Moths / Q000166:cytology*; D011719:Pyrazines / Q000494:pharmacology; D017447:Receptors, Dopamine D1 / Q000819:agonists; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D018491:Dopamine Agonists; D007093:Imidazoles; D008024:Ligands; D011719:Pyrazines; D017447:Receptors, Dopamine D1; D043562:Receptors, G-Protein-Coupled; C017144:coelenterazine; D000331:Aequorin; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins