A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus -: Evidence for Gαi and Gαo coupling to a human leukotriene B4 receptor

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 B-4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [H-3]leukotriene B-4 binding activity (K-d = 3.67 nM). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galpha(i) 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/-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galpha(i1)beta(1)gamma(2) (K-d = 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 Galpha(i1) without Gbeta(1)gamma(2) 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 Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta(1)gamma(2). The BLT1 BV co-expressing Galpha(oA)beta(1)gamma(2) exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galpha(i1)beta(1)gamma(2). Co-expression of other Galpha isoforms such as Galpha(s), Galpha(11), Galpha(14), Galpha(16), Galpha(12), or Galpha(13) 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 Galpha(o) as well as Galpha(i) 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.