A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus

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. 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. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, guanine nucleotide-binding protein (G protein)-coupled receptor; Gα, G protein α-subunit; Gβ1γ2, G protein β1γ2 dimer; Kd, agonist dissociation constant; Ki, agonist inhibition constant; Bmax, relative maximum binding; GTPγS, guanosine 5′-3-O-(thio)triphosphate; Gpp(NH)p, guanosine-5′-(β,γ-imido)triphosphate; Sf9, S. frugiperda 9; LTB4, leukotriene B4 (5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid); BLT1, leukotriene B4 receptor type 1; His-BLT1, His-tagged BLT1; BV, budded virus; TBS, Trisbuffered saline; ELISA, enzyme-linked immunosorbent assay. comprise a superfamily of membrane proteins made up of seven transmembrane segments. GPCRs play an important role in transmembrane signal transduction by binding extracellular ligands such as biogenic amines, peptides, hormones, lipids, and nucleotides (1Gether U. Endocr. Rev. 2000; 21: 90-113Google Scholar). The sensing of exogenous stimuli such as light, odors, and taste is also mediated by this family of GPCRs. Ligand binding activates the heterotrimeric guanine nucleotide-binding proteins (G proteins) on the intracellular face of the plasma membrane (2Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Google Scholar, 3Strange P.G. Biochem. Pharmacol. 1999; 58: 1081-1088Google Scholar, 4Ulloa-Aguirre A. Stanislaus D. Janovick J.A. Conn P.M. Arch. Med. Res. 1999; 30: 420-435Google Scholar, 5Neves S.R. Ram P.T. Iyengar R. Science. 2002; 296: 1636-1639Google Scholar). There is a considerable interest in the investigation of newly identified GPCRs as potential therapeutic targets (6Tate C.G. Grisshammer R. Trends Biotechnol. 1996; 14: 426-430Google Scholar, 7Wilson S. Bergsma D.J. Chambers J.K. Muir A.I. Fantom K.G. Ellis C. Murdock P.R. Herrity N.C. Stadel J.M. Br. J. Pharmacol. 1998; 125: 1387-1392Google Scholar, 8Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Google Scholar). The expression of GPCRs in insect cells with recombinant baculovirus is a common technique (9Bouvier M. Menard L. Dennis M. Marullo S. Curr. Opin. Biotechnol. 1998; 9: 522-527Google Scholar). Sf9 cells, derived from Spodoptera frugiperda, are essentially free of endogenous GPCRs (10Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Google Scholar), providing a low background environment for ligand binding assays. Sf9 cells do endogenously contain certain classes of G proteins that couple to heterologously expressed GPCRs. However, various lines of evidence have made it clear the endogenous Gi-class G proteins exist only at low levels in Sf9 cells (11Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Google Scholar, 12Obosi L.A. Schuette D.G. Europe-Finner G.N. Beadle D.J. Hen R. King L.A. Bermudez I. FEBS Lett. 1996; 381: 233-236Google Scholar). The 5-hydroxytryptamine (10Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Google Scholar, 13Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Google Scholar, 14Francken B.J. Josson K. Lijnen P. Jurzak M. Luyten W.H. Leysen J.E. Mol. Pharmacol. 2000; 57: 1034-1044Google Scholar, 15Brys R. Josson K. Castelli M.P. Jurzak M. Lijnen P. Gommeren W. Leysen J.E. Mol. Pharmacol. 2000; 57: 1132-1141Google Scholar), M2 muscarinic (16Mosser V.A. Amana I.J. Schimerlik M.I. J. Biol. Chem. 2002; 277: 922-931Google Scholar), D2 dopamine (17Cordeaux Y. Nickolls S.A. Flood L.A. Graber S.G. Strange P.G. J. Biol. Chem. 2001; 276: 28667-28675Google Scholar), α2A adrenergic (18Richardson M. Robishaw J.D. J. Biol. Chem. 1999; 274: 13525-13533Google Scholar), and formylmethionylleucylphenylalanine (11Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Google Scholar) receptors have been shown to increase ligand binding activity and/or GTP binding activity in Sf9 cells when co-expressed with Gi-class G proteins. Although Sf9 cells are able to support very high expression levels of exogenous genes (19Possee R.D. Curr. Opin. Biotechnol. 1997; 8: 569-572Google Scholar), the D2 dopamine receptor expressed in Sf9 cells is largely unglycosylated (20Boundy V.A. Lu L. Molinoff P.B. J. Pharmacol. Exp. Ther. 1996; 276: 784-794Google Scholar). In the case of the β2-adrenergic receptor and the formylmethionylleucylphenylalanine receptor, immature and incompletely glycosylated forms of these receptors in the cell membrane fraction of Sf9 cells have also been reported (11Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Google Scholar, 21Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: Scholar, M. H. Dennis M. M. J. Biol. Chem. 1992; 267: Scholar). H. S. P. Marullo S. M. Biotechnol. 1997; Scholar) recovered β2-adrenergic receptor from extracellular baculovirus particles by of β2-adrenergic receptor recombinant baculovirus with a baculovirus the of the human virus type In with been of the recombinant β2-adrenergic receptor was from the particles and were to be with extracellular baculovirus in a budded the β2-adrenergic receptor expressed on the virus particles been glycosylated and was In were agonist should be that β2-adrenergic receptor is a receptor R. K. U. J. Biol. Chem. 1998; 273: Scholar), and Sf9 cells contain the G protein in L.A. Schuette D.G. Europe-Finner G.N. Beadle D.J. Hen R. King L.A. Bermudez I. FEBS Lett. 1996; 381: 233-236Google Scholar). BLT1 is to be the high affinity receptor for leukotriene B4 which is in and been in activity J. 1996; Scholar, Pharmacol. Rev. 1999; Scholar, T. T. T. Arch. Biochem. 2001; Scholar, C. J. S. T. T. Pharmacol. Rev. Scholar). BLT1 is also reported to couple to and G proteins T. T. K. Y. T. 1997; Scholar, R. C. S. S. J. M. Biochem. J. 1998; Scholar) and is not in G protein In a membrane exhibited binding A.J. P. P. J. Pharmacol. Exp. Ther. 1991; Scholar). is also reported that binding to membrane fraction from was by reconstitution of Gi-class G proteins Gαo T. T. O. Y. T. T. J. Biochem. 1999; Scholar), which is in the and system J. J.D. J. 1999; Scholar). Although these that Gαo the coupling of Gαo with BLT1 not been To extracellular baculovirus expression to test GPCR and G protein be reconstituted on the BV, we the expression of human BLT1. The selectivity of BLT1 coupling with other classes of G proteins was also by of this system. was from and were from The was from or were from A recombinant baculovirus expression Y. R.D. H.A. J. Scholar) was by Y. for A J.A. M.I. Robishaw J.D. J. Biol. Chem. 1992; 267: Scholar) was by T. of of at The cDNA for human Gα14, and were by the cDNA and Sf9 cDNA for human BLT1 T. T. K. Y. T. 1997; Scholar) was by a the and a the and and baculovirus expression These were as and the coding the of the and the of BLT1 was by the and and the was cDNA H. T. S. S. T. M. S. H. K. Y. Sci. U. S. A. Scholar) was with and and Gαi1 cDNA H. R. T. T. M. Y. J. Biol. Chem. Scholar) to an endogenous in the coding was with and and cDNA H. T. S. M. H. S. K. T. Science. 1997; 276: Scholar) was with and cDNA M. Y. H. 1999; Scholar) was with and and with to expression was with and with The of the were with cDNA was by a the and and a the and and at the The for human or were at the and The were by To recombinant Sf9 cells were with the for the BLT1, or and for the G protein subunits were with viruses were by in Sf9 The recombinant were and Sf9 cells were in and in a at were infected with the recombinant baculovirus at a of of for of the BV of baculovirus co-expressing receptor and G Sf9 cells were with recombinant at a of of for virus and BV of baculovirus was to the of H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). were at The of Sf9 cells infected with recombinant baculovirus was at for to the was at for The was in and at for to with cell The was at for The used as BV were in or and at of the membrane the cells were in Manning D.R. 2002; Scholar). were by a made with a The were at for and the were at for The membrane were in and at of the BV fraction by was as by H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). of the BV which been in of was of a in The was at for of were from the of the fraction was at for The were with and used for and was as by Scholar), that were not as to proteins expressed on BV were by and to at were for with at were at with the in The were in and were with with and for with or as the were with with and with were to BV or membrane were to the of a enzyme-linked immunosorbent at were and with TBS, for at with for at were with with The to was with the by the of The was by and at were a Ligand ligand binding was in a of of binding T. T. K. Y. T. 1997; Scholar, K. T. T. T. Biochem. J. 1999; Scholar) the BV fraction and To test the of on binding to the BV of was in the binding with various of The were at for The was by of the The were with of binding and and the was in a The were with the BV were in A. M. S. M. 1999; Scholar) with and BV were with and with or without at for The was by The were with of and and the was in a The BV fraction was recovered from the of Sf9 cells infected with His-tagged BLT1 recombinant BLT1 by with was in the BV fraction with recombinant virus cDNA The expression The amount of in the BV fraction at was that of the membrane fraction from Sf9 binding activity was also observed in the BV and membrane from Sf9 cells that been infected with recombinant baculovirus The maximum binding activity was at which was in the BV the membrane binding activity was in the BV or membrane fraction from Sf9 cells infected with type To BLT1 is expressed on virus or cell the BV fraction from the of Sf9 cells infected with BLT1 recombinant baculovirus was by in shown in was to with baculovirus The expression and of the of BLT1 in are with reported β2-adrenergic receptor expression on extracellular budded virus H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). BLT1 is in this case to be expressed on the budded The affinity for the of BLT1 expressed on BV to be low (Kd = 3.67 with that of the membrane fraction of cells BLT1 (Kd = 0.17 T. T. K. Y. T. 1997; Scholar). This is to be due to the low level of the Gi-class G protein in Sf9 cells, which is to couple to BLT1 for high affinity of to BLT1 expressed on membrane and BV affinity affinity of of in a of and G for protein coupling on BV, we recombinant the the as well as the G protein and and the G protein subunits and were expressed on BV in various of BV fraction was The amount of expressed receptor or of G protein was by the was for BLT1 in the and did not the we without BLT1, the from the is with apparent of and were in BV. did not observed the to the of the BLT1 in this system in the of G The amount of receptor expression with of various G protein subunits in the membrane and BV This was with an was with in the type BV G protein expressed an amount on levels of BLT1 on BV co-expressing G protein BV His-tagged BLT1 with or without various G protein subunits were to with an = of of protein were and type the BV subunits and the budded type Ligand of and G BV recovered from with recombinant virus a binding for a and In the binding of to BLT1 expressed on BV or membrane contain high and low affinity and The and from the are shown in I. shown in the ligand binding were in the Sf9 membrane and BV the receptor BLT1, the and of the high affinity binding were and pmol/mg of and of the low affinity were 3.67 and pmol/mg of = The to the of the BLT1 did not ligand binding not There was significant specific binding in the BV fraction from the of Sf9 cells infected with the type To the effects of G protein on agonist the Sf9 membrane and BV co-expressing receptor and G proteins were used for the binding shown in the of the membrane fraction co-expressing with BLT1 and Gαi1 was pmol/mg of which is the membrane fraction BLT1 The co-expression of Gβ1γ2 subunits with BLT1 also the high affinity by in the membrane the BV the BLT1 and Gαi1 exhibited a relatively of high affinity binding = pmol/mg of protein). of the G protein the receptor to the high affinity with a of 0.17 the of the high affinity receptor with the receptor expressed A GTP Gpp(NH)p, which is not by the activity of the Gα was used to protein binding to BV and providing evidence for the protein coupling the other the BLT1 BV, BLT1 with Gαi1 BV, and BLT1 with Gβ1γ2 BV were not in ligand binding by the of These results that the co-expression of the receptor and heterotrimeric G protein to GPCR-G protein on BV. To the of the G protein of various classes on ligand Sf9 cells were with various of recombinant baculovirus classes of G protein subunits with Gβ1γ2 in and BV fraction was by and of the expressed proteins was by The expression levels of by were the BV G proteins not specific were by in the BV fraction recovered from the of Sf9 cells infected with type The ligand binding of these BV were shown in an increase of high affinity binding was observed in the BV with and the heterotrimeric G protein co-expression of heterotrimeric G protein with also led to an increase in the high affinity binding the other BV co-expressing with other classes of G protein and exhibited a low affinity ligand binding to that of BV shown in the of BV co-expressing and Gαi1β1γ2 or GαoAβ1γ2 in the case of the high affinity was to that of BV co-expressing other classes of heterotrimeric G proteins in the low affinity These results that the expressed receptors in the BV were functionally coupled to Gαi1β1γ2 and binding to the BV co-expressing BLT1 and various classes of Gα with of BV co-expressing BLT1 and various classes of Gα subunits with Gβ1γ2 are shown = binding to budded viruses BLT1 and BLT1 and Gαi1β1γ2 BLT1 and GαoAβ1γ2 BLT1 and BLT1 and BLT1 and BLT1 and BLT1 and and BLT1 were In of BV BLT1 or of that of co-expressing BLT1 with Gαi1β1γ2 or GαoAβ1γ2 was other BV were were to a The and were derived for and are in of to BLT1 expressed on BV affinity affinity of of in a To the coupling of G proteins to BLT1, the of binding by in the or of was the various BLT1 BV co-expressing of heterotrimeric G proteins that the agonist the apparent affinity of binding it also the affinity of binding J. Biol. Chem. 1992; 267: Scholar, S.R. J. Biol. Chem. 1998; 273: Scholar). The binding of was by in BV co-expressing BLT1 and or heterotrimeric G proteins. for BLT1 BV co-expressed with Gαi1β1γ2 or GαoAβ1γ2 were to other in the or of the ligand and The for the of BLT1 BV co-expressing Gαi1β1γ2 or GαoAβ1γ2 were and in the of LTB4, and and in the of LTB4, These results that BLT1 couples to as well as heterotrimeric G proteins in BV. the BLT1 BV co-expressing G did not exhibit a significant on the of binding by Although the binding of was by in BLT1 BV co-expressed with heterotrimeric G the effects of were that for BLT1 BV co-expressed with Gi-class G protein The for binding on BLT1 BV co-expressed with G proteins and were by and in the of The BLT1 BV co-expressing G protein also a in binding by and The for the of BLT1 BV co-expressed with and were by and in the of of Sf9 cells the recombinant membrane proteins functionally in the particles H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). this by a co-expression system with GPCR and G proteins to the high affinity In we observed that the co-expression of G protein subunits GPCR-G protein coupling in of ligand Sf9 cells infected with baculovirus released BV on which the BLT1 receptor was The BV fraction exhibited ligand binding activity the membrane the membrane fraction an amount of receptor These results are in with a that β2-adrenergic receptor expression on extracellular virus H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). β2-adrenergic receptor expressed on the BV of baculovirus exhibited specific ligand binding activity H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). receptor is to couple to the of G protein R. K. U. J. Biol. Chem. 1998; 273: Scholar). is in Sf9 cells, the recovered baculovirus G protein such that evidence that the high affinity receptor was expressed in BV H. S. P. Marullo S. M. Biotechnol. 1997; Scholar). BLT1 was expressed the BV fraction exhibited a of high affinity and a large of low affinity ligand binding The of to BLT1 expressed on BV in the high affinity was which is to that of human BLT1 expressed in cells reported T. T. K. Y. T. 1997; Scholar). The high affinity and binding are thought to receptors coupled to G the low affinity In Sf9 cells, various G protein are expressed in on the of G protein L.A. Schuette D.G. Europe-Finner G.N. Beadle D.J. Hen R. King L.A. Bermudez I. FEBS Lett. 1996; 381: 233-236Google Scholar, D. C. Arch. Pharmacol. 1997; Scholar). The of the high affinity binding observed in BV BLT1 a receptor coupled to endogenous G proteins. In this we Sf9 cells with recombinant the for BLT1 and heterotrimeric G protein The Gβ1γ2 combination used is of with classes of the G protein A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Google Scholar, J. Biol. Chem. Scholar). In the expression of the receptor and G protein subunits was with The expression level of in recovered BV well to the amount of recombinant baculovirus used to The of the receptors expressed in the membrane fraction from Sf9 cells with BLT1 and Gαi1β1γ2 recombinant exhibited high affinity ligand binding a and In the membrane co-expression of BLT1 and Gαi1β1γ2 high affinity ligand binding with BLT1 co-expression of BLT1 with Gαi1 led to a increase in the high affinity binding with that of BLT1 by subunits have been reported to be in Sf9 cells D. C. Arch. Pharmacol. 1997; Scholar), the endogenous subunits of Sf9 cells well be to the membrane fraction co-expressing receptor and the Gα and the with exogenous The BV released from the cells also exhibited high affinity for ligand binding with the binding affinity observed in expression T. T. K. Y. T. 1997; Scholar) and the BV co-expression of BLT1 and Gαi1 without in only a increase in the high affinity binding and BV a amount of the endogenous was for BV to Gαi1 and with BLT1 to maximum binding These results are with the that is for protein A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Google Scholar, Y. Nickolls S.A. Flood L.A. Graber S.G. Strange P.G. J. Biol. Chem. 2001; 276: 28667-28675Google Scholar, M. Robishaw J.D. J. Biol. Chem. 1999; 274: 13525-13533Google Scholar, J. Biol. Chem. Scholar). The in the of high affinity ligand binding on BV on the expression of exogenous G protein these a BV co-expression system a for the reconstitution of GPCR and G proteins the background of the endogenous G proteins is in the cell membrane The Sf9 cell expression system also been used to the coupling of receptors G proteins B.J. Josson K. Lijnen P. Jurzak M. Luyten W.H. Leysen J.E. Mol. Pharmacol. 2000; 57: 1034-1044Google Scholar, Y. Nickolls S.A. Flood L.A. Graber S.G. Strange P.G. J. Biol. Chem. 2001; 276: 28667-28675Google Scholar). BLT1 BV co-expressed with GαoAβ1γ2 as well as Gαi1β1γ2 exhibited high affinity ligand binding and also binding These that GαoAβ1γ2 is able to couple to BLT1 and that of the receptor led to a with a also that receptor from was able to couple with exogenous Gi-class G Gαo T. T. O. Y. T. T. J. Biochem. 1999; Scholar). The of the BLT1 Gαo to be Gαo is reported to be expressed only in and J. J.D. J. 1999; Scholar) and receptor BLT1 in and also in and at low levels T. T. K. Y. T. 1997; Scholar). BLT1 expression was reported in cells M. Biochem. J. 2002; Scholar). A specific for BLT1 of in cells M. Biochem. J. 2002; Scholar). BLT1 expression was also in the of and other in a of human and The of These are of a for BLT1 in the system. The co-expression of BLT1 with any of the other G proteins Gα11, Gα14, Gα16, Gα12, and Gα13 with Gβ1γ2 in significant on ligand binding A the did not couple to BLT1 in the T. T. K. Y. T. 1997; Scholar). The Gα16, not Gα11, was to be coupled to BLT1 in R. C. S. S. J. M. Biochem. J. 1998; Scholar). the BLT1 BV co-expressing heterotrimeric G we observed a in the of binding by in the or of ligand The of binding was Gα11, which was with the R. C. S. S. J. M. Biochem. J. 1998; Scholar). the G it is that Gα13 also BLT1 such as of Although and Gα13 to the of G and effects S.R. Ram P.T. Iyengar R. Science. 2002; 296: 1636-1639Google Scholar). Gα13 been reported to with and a guanine for the which activates T. M.J. P.M. Science. 1998; Scholar, M.J. T. W. Science. 1998; Scholar). a of such as and by the of the S. A. 2002; Scholar). In BLT1 BV Gα12, with Gβ1γ2 exhibited binding by and that BLT1 expressed on BV the of protein coupling This this expression system should prove highly useful for the of coupling of newly identified receptors to various classes of Gα the receptors and were reported to have virus virus particles receptors were to an biosensor and were in and human binding L. J. Sci. U. S. A. 2000; Scholar). it was to the baculovirus BV on a with a of K. K. M. K. and T. a that led to the of the virus In a baculovirus expression the GPCR expressed on BV for at at not in of This system is to have the other expression in of of a These the of recombinant viruses various G protein the reconstitution of high affinity receptors by of recombinant as and the low background of the GPCR in Sf9 This system is also to be of considerable in to the of GPCR and G proteins as well as other such as the of G protein K. and T. for of G protein recombinant and K. T. H. M. and Y. for are to K. for the