title: Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors.
abstract: We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha16. We now show that, compared with Galpha16, the use of chimeric Galphaq subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect Gi-coupled GPCRs into a Gq-type signal transduction pathway. We coexpressed human Gi-coupled GPCRs, G protein alpha subunits (either a chimeric Galphaq or Galpha16), and the calcium-sensitive reporter protein aequorin in Sf9 cells using a nonlytic protein expression system, and measured agonist-induced intracellular calcium flux using a luminometer. Three of the GPCRs (serotonin 1A, 1D, and dopamine D2) were functionally redirected into a Gq-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha16. We determined agonist concentration-response relationships for all three receptors, which yielded EC50 values comparable with those achieved in mammalian cell-based assay systems. However, three other Gi-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha16. Possible reasons and solutions for this result are discussed.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, Cambs, UK.
authors.forename: Peter J K
authors.initials: PJ
authors.lastname: Knight
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Grigliatti
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007313:Insecta; D011719:Pyrazines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry
chemical_list: D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D007093:Imidazoles; D011719:Pyrazines; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C017144:coelenterazine; D010738:Type C Phospholipases