Chimeric G Proteins Extend the Range of Insect Cell-Based Functional Assays for Human G Protein-Coupled Receptors

Publisher Website
Google Scholar
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 Gα16. We now show that, compared with Gα16, the use of chimeric Gαq 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 α subunits (either a chimeric Gαq or Gα16), 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 Gα16. 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 κ1 and δ1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Gα16. Possible reasons and solutions for this result are discussed.