A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao 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 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 Galphai 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 Galphai1beta1gamma2 (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 Galphai1 without Gbeta1gamma2 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 Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 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 Galphao as well as Galphai 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.
Masuda
Kazuyuki
K
Laboratory for Systems Biology and Medicine, The University of Tokyo, Japan.
Itoh
Hiroshi
H
Sakihama
Toshiko
T
Akiyama
Chiyuki
C
Takahashi
Kazuaki
K
Fukuda
Rie
R
Yokomizo
Takehiko
T
Shimizu
Takao
T
Kodama
Tatsuhiko
T
Hamakubo
Takao
T
D016367:Baculoviridae; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D018102:Receptors, Leukotriene B4 / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D015398:Signal Transduction; D014771:Virion
D008024:Ligands; D018102:Receptors, Leukotriene B4; D011994:Recombinant Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go