Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors

We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha(16). We now show that, compared with Galpha(16), the use of chimeric Galpha(q) subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect G(i)-coupled GPCRs into a G(i)-type signal transduction pathway. We coexpressed human G(i)-coupled GPCRs, G protein a subunits (either a chimeric Galpha(q) or Galpha(16), 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 G(q)-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha(16). 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 G(i)-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha(16). Possible reasons and solutions for this result are discussed.