A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus EVIDENCE FOR Gαi AND Gαo COUPLING TO A HUMAN LEUKOTRIENE B4 RECEPTOR

Abstract To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Gαi isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5′-(β,γ-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Gαi1β1γ2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Gαi1 without Gβ1γ2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Gα subunits were combinatorially expressed in BV with BLT1 and Gβ1γ2. The BLT1 BV co-expressing GαoAβ1γ2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5′-3-O-(thio)triphosphate binding to Gαi1β1γ2. Co-expression of other Gα isoforms such as Gαs, Gα11, Gα14, Gα16, Gα12, or Gα13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Gαo as well as Gαi couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.