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.

G protein-coupled receptors (GPCRs) 1 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 (1). 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 (2)(3)(4)(5). There is a considerable interest in the investigation of newly identified GPCRs as potential therapeutic targets (6 -8).
Although Sf9 cells are able to support very high expression levels of exogenous genes (19), the D 2 dopamine receptor expressed in Sf9 cells is largely unglycosylated (20). In the case of the ␤ 2 -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 (11,21,22).
Loisel et al. (23) successfully recovered functional ␤ 2 -adrenergic receptor from extracellular baculovirus particles by coinfection of ␤ 2 -adrenergic receptor recombinant baculovirus with a baculovirus encoding the Pr55 Gag product of the human immunodeficiency virus type 1. In contrast with what had been expected, most of the recombinant ␤ 2 -adrenergic receptor was excluded from the Gag particles and were found instead to be associated with extracellular baculovirus (BV) in a budded form. Surprisingly, the ␤ 2 -adrenergic receptor expressed on the virus particles had been glycosylated and was biologically active. In addition, post-translational modifications were found upon agonist stimulation. It should be noted here that ␤ 2adrenergic receptor is a G s -coupling receptor (24), and Sf9 cells contain the G s class G protein in some abundance (12).
BLT1 is known to be the high affinity receptor for leukotriene B 4 (LTB 4 ), which is found mainly in leukocytes and has been implicated in inflammatory activity (25)(26)(27)(28). BLT1 is also reported to couple to G i -and G q -class G proteins (29,30) and is not involved in G s -class G protein signaling. In a previous report, guinea pig brain membrane exhibited LTB 4 -specific binding (31). It is also reported that LTB 4 binding to partially purified membrane fraction from porcine leukocytes was in- . The BV and membrane fractions were collected at various times after infection. a, the BV fractions were collected from Sf9 cells infected with His-BLT1 recombinant (q) or wild type (wt, f) baculovirus. The membrane fractions were also prepared from Sf9 cells infected with His-BLT1 recombinant (E) or wild type virus (Ⅺ). Samples (5 g of protein) were used to detect the immunoreactivity. b, 1 g of protein of the BV or membrane fractions was incubated with 0.25 nM [ 3 H]LTB 4 , and bound radioactivity was detected. Data represent the mean Ϯ S.D. (n ϭ 4). creased by reconstitution of G i -class G proteins including G␣ o (32), which is found in the brain and nervous system (33). Although these reports suggest that G␣ o mediates LTB 4 signaling, the coupling of G␣ o with BLT1 has not been investigated intensively. To exploit extracellular baculovirus expression to test whether GPCR and G protein could be reconstituted on the BV, we studied the expression of human BLT1. The possible selectivity of BLT1 coupling with other classes of G proteins was also studied by means of this system.  lovirus expression vectors (Invitrogen). These were designated as pBB4.5-BLT1 and pBBH2A-BLT1, respectively. For pBBH2A-BLT1, the residual coding sequence between the enterokinase restriction site of the vector and the first Met of BLT1 was deleted by site-directed mutagenesis using the primers 5Ј-CTGTACGACGATGACGATAAGAT-GAACACTACATCTTCTGCAGC-3Ј and 5Ј-GCTGCAGAAGATGTAGT-GTTCATCTTATCGTCATCGTCGTACAG-3Ј, and the resulting plasmid was designated pBBH2-BLT1. Rat G␣ s cDNA (36) was digested with NcoI and HindIII and inserted into pBlueBacIII (Invitrogen). Rat G␣ i1 cDNA (37) mutated to lack an endogenous NcoI site in the coding region was digested with NcoI and HindIII and inserted into pBlueBacIII. Mouse G␣ 11 cDNA (38) was digested with HindIII and inserted into pBlueBacIII. Rat G␣ 12 cDNA (39) was digested with Bsu36I and BglII and treated with Klenow fragment to generate blunt ends. Baculovirus expression vector pAcYMI was digested with BamHI and treated with Klenow fragment. The blunt-end fragments of the vector were ligated with G␣ 12 cDNA. Bovine G␤ 1 cDNA was amplified by PCR using a 5Ј sense primer containing the SmaI and XhoI sites and a 3Ј antisense primer containing the SmaI and EcoRI sites and inserted into pAcYMI at the SmaI site. The cDNAs for human G␣ oA , Ga 13 , Ga 14 , or Ga 16 were subcloned individually into pBlueBac4.5 at the KpnI and XbaI sites. The nucleotide sequences were verified by DNA sequencing. To generate recombinant viruses, Sf9 cells were co-transfected with the vector for either the BLT1, G␣ oA , Ga 13 , Ga 14 , or Ga 16 and Bac-N-Blue viral DNA (Invitrogen). Other vectors for the G protein subunits were cotransfected individually with BaculoGold viral DNA (BD Biosciences). All viruses were produced by homologous recombination in Sf9 cells. The recombinant baculoviruses thus obtained were plaque-purified and amplified. Sf9 cells were cultured in Grace's supplemented media (Invitrogen) containing 10% fetal calf serum, 0.1% Pluronic F-68 (Invitro-gen), 100 international unit/ml penicillin and 100 g/ml streptomycin in a 1-liter spinner flask at 27°C. Cells (2 ϫ 10 6 cells/ml) were infected with the recombinant baculovirus at a multiplicity of infection of 5 for 72-120 h. For production of the BV of baculovirus co-expressing receptor and G proteins, Sf9 cells (2 ϫ 10 6 cells/ml) were co-infected with recombinant baculoviruses at a multiplicity of infection of 2 for each virus and harvested 72 h after infection.

Reagents-LTB
Viral Particle Preparation-The BV of baculovirus was isolated basically according to the method of Loisel et al. (23). All procedures were carried out at 0 -4°C unless stated otherwise. The culture medium of Sf9 cells infected with recombinant baculovirus was centrifuged at 1,000 ϫ g for 15 min to separate cells. Then the supernatant was centrifuged at 40,000 ϫ g for 30 min. The pellet was resuspended in phosphate-buffered saline and centrifuged at 1,000 ϫ g for 15 min to eliminate possible contamination with cell debris. The supernatant was again centrifuged at 40,000 ϫ g for 30 min. The precipitates, used as BV fractions, were resuspended in phosphate-buffered saline or assay buffer and stored at 4°C until use.
For preparation of the membrane fraction, the cells were resuspended in HE/PI buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 10 g/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride) (40). Cells were broken open by a path made with a 26-gauge needle inserted 15 times. The homogenates were centrifuged at 1,000 ϫ g for 5 min, and the supernatants were further centrifuged at 20,000 ϫ g for 30 min. The precipitates (the membrane fractions) were suspended in HE/PI buffer and stored at Ϫ20°C until use.
Sucrose Density Gradient Sedimentation-Fractionation of the BV fraction by sucrose density gradient sedimentation was performed as described by Loisel et al. (23). Briefly, 600 g of the BV fraction, which had been suspended in 1.2 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) was layered onto 36 ml of a linear 25-56% (w/v) sucrose gradient in TE buffer. The sample was centrifuged at 10,000 ϫ g for 90 min. Fractions of 1.5 ml were collected from the top of the tube. Then each fraction was centrifuged at 20,000 ϫ g for 45 min. The precipitates were resuspended with phosphate-buffered saline and used for Western blot analysis.
Electrophoresis and Immunoblotting-SDS-PAGE was carried out as described by Laemmli (41), except that samples were not heat-treated so as to minimize aggregation. For immunoblotting, proteins expressed on BV were resolved by SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences) electrophoretically at 20 V overnight. Membranes were incubated for 1 h with Block Ace (Snow Brand Milk Products, Japan) at room temperature. Membranes were then incubated overnight at 4°C with the primary antibody in Tris-buffered saline (TBS; 137 mM NaCl, 3 mM KCl, 25 mM Tris-HCl, pH 7.4). The following antibodies were used: monoclonal mouse anti-baculoviral glycoprotein gp64 (established in our laboratory); monoclonal mouse anti-polyhistidine (Sigma); polyclonal rabbit anti-G␣ s (K-20 sc-823), anti-G␣ oA (K-20 sc-387), anti-G␣ 11  The membrane fraction and BV fraction were prepared from infected cells and culture supernatants, respectively. Because of the difference of expression level of BLT1 in each sample the membrane or BV fraction was loaded in panel a as follows: the membrane fraction expressing His-BLT1 and G␣ i1 ␤ 1 ␥ 2 (30 g), His-BLT1 and G␣ i1 (30 g), His-BLT1 and G␤ 1 ␥ 2 (30 g), His-BLT1 alone (3 g) from cells infected with wild type (wt) baculovirus (30 g); the BV fraction expressing His-BLT1 and G␣ i1 ␤ 1 ␥ 2 (50 g), His-BLT1 and G␣ i1 (50 g), His-BLT1 and G␤ 1 ␥ 2 (50 g), His-BLT1 alone (5 g), and wild type baculovirus (50 g).   4. Expression levels of BLT1 on BV co-expressing G protein subunits. BV samples expressing His-tagged BLT1 with or without various G protein subunits were subjected to ELISA with an anti-His antibody (n ϭ 4, mean Ϯ S.D.). Samples of 3 g of protein were used. ␤␥ and wild type (wt) represent the BV expressing G␤ 1 plus G␥ 2 subunits and the budded wild type baculovirus, respectively. OD, optical density.
Enzyme-linked Immunosorbent Assay-The BV or membrane fractions were adsorbed to the wells of a 96-well enzyme-linked immunosorbent assay (ELISA) plate (Asahi Techno Glass, Tokyo, Japan) overnight at 4°C. Wells were washed and blocked with TBS, pH 7.4, containing 40% Block Ace for 1 h at room temperature. After incubation with mouse monoclonal anti-polyhistidine antibody for at least 1 h, wells were washed 3 times with TBS with 0.05% Tween 20. The anti-polyhistidine antibody bound to wells was detected with the horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody followed by the addition of substrate (tetramethylbenzidine). The reaction was terminated by acid, and absorbances at 450 nm were quantitated using a 96-well plate reader.
Ligand Binding Assay-The radiolabeled ligand binding assay was carried out in a total of 200 l of binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 10 mM NaCl, 0.1% fatty acid-free bovine serum albumin) (29,42) 4 binding to the BV fractions, 0.02% (w/v) of saponin was added in the binding buffer with various amounts of Gpp(NH)p. The assay mixtures were incubated at room temperature for 1 h. The incubation was terminated by filtration of the assay mix-

FIG. 5. [ 3 H]LTB 4 binding to the membrane or BV fractions from Sf9 cells co-infected with recombinant baculoviruses encoding BLT1 and G protein subunits.
Binding isotherms and Scatchard analyses of various membrane or BV fractions co-expressing BLT1 and G protein subunits are shown. Values are represented as the means Ϯ S.E. (n ϭ 4). The membrane or BV fractions (1 g each) expressing BLT1 and G␣ i1 ␤ 1 ␥ 2 (f), BLT1 and G␣ i1 (‚), BLT1 and G␤ 1 ␥ 2 (ƒ), and BLT1 alone (q) were analyzed. Isotherms and Scatchard analyses were best fitted to a two-binding-site model using nonlinear analysis. In the Scatchard plot, only the high affinity binding sites from each membrane or BV fractions are designated as indicated on the figure panels for BLT1 and G␣ i1 ␤ 1 ␥ 2 , BLT1 and G␣ i1 , BLT1 and G␤ 1 ␥ 2 , and BLT1 alone. The K d and B max values were derived from each individual experiment, and the means Ϯ S.E. are summarized in Table I. tures through GF/C glass fiber filters (PerkinElmer Life Sciences). The filters were washed with ϳ2 ml of ice-cold binding buffer and dried, and the remaining radioactivity was measured in a Top-count microplate scintillation counter (PerkinElmer Life Sciences). The data were analyzed with the program GraphPad Prism (GraphPad Software, San Diego, CA).

Expression of BLT1 on Budded Baculovirus
The BV fraction was recovered from the culture medium of Sf9 cells infected with His-tagged BLT1 (His-BLT1) recombinant baculovirus. BLT1 expression, detected by ELISA with anti-His antibody, was seen in the BV fraction 48 h after infection with recombinant virus carrying His-BLT1 cDNA (Fig. 1a). The His-BLT1 expression reached its peak 72 h after infection. The amount of His-BLT1 in the BV fraction at 72 h was 1.4-fold higher than that of the membrane fraction from His-BLT1-infected Sf9 cells. [ 3 H]LTB 4 binding activity was also observed in both the BV and membrane fractions prepared from Sf9 cells that had been infected with His-BLT1 recombinant baculovirus (Fig. 1b). The maximum binding activity was reached at 72 h after infection, which was about 3-fold higher in the BV than the membrane fraction. No binding activity was detected in the BV or membrane fraction collected from Sf9 cells infected with wild type baculovirus.
To clarify whether BLT1 is expressed on virus or cell debris, the BV fraction from the culture medium of Sf9 cells infected with BLT1 recombinant baculovirus was analyzed by ultracentrifugation in sucrose density gradients. As shown in Fig. 2, His-BLT1 was found to cosediment with baculovirus glycoprotein gp64. The expression time course and properties of the sedimentation of BLT1 in culture supernatant are compatible with previously reported ␤ 2 -adrenergic receptor expression on extracellular budded virus (23). Thus, BLT1 is likewise deduced in this case to be expressed on the budded baculovirus. The affinity for the [ 3 H]LTB 4 of BLT1 expressed on BV seems to be low (K d ϭ 3.67 nM, Table I) compared with that of the membrane fraction of Cos-7 cells expressing BLT1 (K d ϭ 0.17 nM) (29). This is considered to be due to the low level of the G i -class G protein in Sf9 cells, which is needed to couple to BLT1 for high affinity binding.

Reconstitution of BLT1-G Protein Coupling on Budded Virus
Expression of Receptor and Heterotrimeric G Protein-To check for BLT1-G protein coupling on BV, we prepared recombinant baculoviruses containing the cDNAs encoding the His-BLT1 as well as the G protein ␣ i1 , ␤ 1 , and ␥ 2 subunits. His-BLT1 and the G protein subunits G␣ i1 , G␤ 1 , and G␥ 2 were expressed on BV in various combinations. After 72 h of infection, each BV fraction was collected. The amount of expressed receptor or each subunit of G protein was assessed by Western blotting (Fig. 3). When the sample was heat-treated for SDS-PAGE, BLT1 stacked in the stacking gel and did not enter the separating gel. Therefore, we performed SDS-PAGE analysis without heat treatment. For BLT1, the deduced molecular mass from the amino acid sequence is 37.6 kDa. Using anti-His antibody immunoblotting, three bands, with apparent molecu-lar masses of 36.6, 35.5, and 34.8 kDa, were detected in His-BLT1-expressing BV. We did not observed the bands corresponding to the dimmers of the BLT1 in this system even in the presence of G protein. The amount of receptor expression decreased with co-infection of various G protein subunits in both the membrane and BV fractions (Fig. 3a). This observation was corroborated with ELISA measurement using an anti-His antibody (Fig. 4). No immunoreactive band was detected with anti-His antibody in the wild type BV sample. Each G protein subunit expressed an apparently similar amount on BV.
Ligand Binding Assay of Reconstituted Receptor and G Protein-The BV fractions recovered from culture medium 72 h after infection with His-BLT1 recombinant virus displayed a dose-dependent binding for [ 3 H]LTB 4 (Fig. 5, a and b). In Scatchard plot analysis, the binding properties of [ 3 H]LTB 4 to BLT1 expressed on BV or membrane fractions contain both high and low affinity sites (Fig. 5, c and d). The K d and B max values calculated from the Scatchard plot are shown in Table I. As shown in Table I, the ligand binding properties were similar in the Sf9 membrane and BV fractions expressing the receptor alone. For BV-expressed BLT1, the mean K d and B max values of the high affinity binding sites were 0.13 nM and 0.64 pmol/mg of protein, and those of the low affinity sites were 3.67 nM and 27.3 pmol/mg of protein, respectively (n ϭ 4). The His hexapeptide fused to the N terminus of the BLT1 did not affect ligand binding (data not shown). There was no significant specific [ 3 H]LTB 4 binding in the BV fraction prepared from the culture medium of Sf9 cells infected with the wild type baculovirus.
To estimate the effects of G protein on agonist affinity, the Sf9 membrane and BV fractions co-expressing receptor and G proteins were used for the [ 3 H]LTB 4 binding experiments (Fig.  5). As shown in Table I, the B max value of the membrane fraction co-expressing with BLT1 and G␣ i1 was 3.78 pmol/mg of protein, which is 2.8-fold greater than the membrane fraction expressing BLT1 alone. The co-expression of G␤ 1 ␥ 2 subunits with BLT1 also increased the high affinity site by 1.5-fold in the membrane fraction. For the BV fraction, the sample coexpressing BLT1 and G␣ i1 exhibited a relatively small number of high affinity binding sites (B max ϭ 1.92 pmol/mg of protein). Reconstitution of the G protein heterotrimer (G␣ i1 ␤ 1 ␥ 2 ) switched almost all the entire receptor population to the high affinity state with a K d value of 0.17 nM, increasing the B max value of the high affinity receptor 9.5-fold compared with the receptor expressed alone. Budded virus expressing BLT1 and G␣ i1 ␤ 1 ␥ 2 , BLT1 and G␣ i1 , BLT1 and G␤ 1 ␥ 2 , and BLT1 alone (as indicated in the figure) were analyzed. Statistical analysis was performed using Student's t test. Significant inhibition by Gpp(NH)p is indicated as p Ͻ 0.001 (*).

The Effect of Gpp(NH)p on Ligand Binding to BV Expressing BLT1 and G Proteins
A GTP analogue, Gpp(NH)p, which is not hydrolyzed by the GTPase activity of the G␣ protein, was used to delineate receptor-G protein coupling. Gpp(NH)p decreased [ 3 H]LTB 4 binding to BV expressing His-BLT1 and G␣ i1 ␤ 1 ␥ 2 , providing evidence for the receptor-G protein coupling (Fig. 6). On the other hand, the BLT1 BV, BLT1 with G␣ i1 BV, and BLT1 with G␤ 1 ␥ 2 BV were not significantly affected in ligand binding by the addition of Gpp(NH)p. These results suggest that the co-expression of the receptor and heterotrimeric G protein reconstitute to functional GPCR-G protein complex on BV.

The Effect of the Co-expression of Various Classes of G Protein on the [ 3 H]Leukotriene B 4 Binding Properties of BLT1 Expressed on BV
To compare the effect of the G protein ␣ subunit of various classes on ligand binding, Sf9 cells were co-infected with various combinations of recombinant baculovirus encoding several different classes of G protein subunits (␣ with G␤ 1 ␥ 2 in every case) and His-BLT1. Each BV fraction was collected by centrifugation, and each of the expressed proteins was detected by Western blotting (Fig. 7). The expression levels of His-BLT1 assessed by ELISA were similar among the BV fractions expressing different G proteins (data not shown). No specific signals were detected by Western blotting in the BV fraction recovered from the culture medium of Sf9 cells infected with wild type baculovirus. The ligand binding properties of these BV fractions were compared. As shown in Fig. 8, an increase of high affinity binding sites was observed in the BV co-infected with His-BLT1 and the G i heterotrimeric G protein (G␣ i1 ␤ 1 ␥ 2 ). Furthermore, co-expression of G o heterotrimeric G protein with His-BLT1 (G␣ oA ␤ 1 ␥ 2 ) also led to an increase in the high affinity binding sites. On the other hand, BV fractions co-expressing His-BLT1 with other classes of G protein heterotrimers (G s , G 11 , G 14 , G 16 , G 12 , and G 13 ) exhibited a low affinity ligand binding similar to that of BV expressing His-BLT1 alone. As shown in Table II, the B max value of BV co-expressing His-BLT1 and G␣ i1 ␤ 1 ␥ 2 or G␣ oA ␤ 1 ␥ 2 in the case of the high affinity sites was similar to that of BV co-expressing other classes of heterotrimeric G proteins in the low affinity sites. These results indicate that almost all the expressed receptors in the former two BV fractions were functionally coupled to G␣ i1 ␤ 1 ␥ 2 and G␣ oA ␤ 1 ␥ 2 .

[ 35 S]GTP␥S Binding of Heterotrimeric G Proteins Expressed in Budded Virus
To further examine the selective coupling of G proteins to BLT1, the displacement of [ 35 S]GTP␥S binding by GDP in the presence or absence of 100 nM LTB 4 was compared among the various BLT1 BV co-expressing different combinations of heterotrimeric G proteins (Fig. 9). Previous studies demonstrate that the agonist increases the apparent affinity of [ 35 S]GTP␥S binding sites; it also decreases the affinity of GDP binding sites (44,45). The binding of [ 35 S]GTP␥S was greatly increased by LTB 4 in BV co-expressing BLT1 and G i or G o heterotrimeric G proteins. Displacement curves for BLT1 BV co-expressed with G␣ i1 ␤ 1 ␥ 2 or G␣ oA ␤ 1 ␥ 2 were similar to each other both in the presence or absence of the ligand (Fig. 9, b and c). The IC 50 values for the GDP of BLT1 BV co-expressing G␣ i1 ␤ 1 ␥ 2 or G␣ oA ␤ 1 ␥ 2 were 0.25 and 0.80 M in the absence of LTB 4 , and 7.58 and 12.45 M in the presence of LTB 4 , respectively. These results show that BLT1 couples to G o as well as G i heterotrimeric G proteins in BV.
For the BLT1 BV co-expressing G s -class G proteins, LTB 4 did not exhibit a significant effect on the displacement of [ 35 S]GTP␥S binding by GDP (Fig. 9a). Although the binding of [ 35 S]GTP␥S was apparently induced by LTB 4 in BLT1 BV co-expressed with G q -class heterotrimeric G proteins, the effects of LTB 4 were much less than that for BLT1 BV co-expressed with G i -class G protein (Fig. 9, d-f). The GDP IC 50 values for [ 35 S]GTP␥S binding on BLT1 BV co-expressed with G q -class G proteins (G␣ 11 ␤ 1 ␥ 2 , G␣ 14 ␤ 1 ␥ 2 , and G␣ 16 ␤ 1 ␥ 2 ) were increased by 2.3-, 2.4-, and 3.1-fold, respectively, in the presence of LTB 4 . The BLT1 BV co-expressing G 12/13 -class G protein also showed a slight difference in [ 35 S]GTP␥S binding by LTB 4 (Fig. 9, g and h). The IC 50 values for the GDP of BLT1 BV co-expressed with G␣ 12 ␤ 1 ␥ 2 and G␣ 13 ␤ 1 ␥ 2 were increased by 2.3-and 2.6-fold, respectively, in the presence of LTB 4 .

DISCUSSION
Baculovirus infection of Sf9 cells renders the recombinant membrane proteins functionally embedded in the viral parti-

H]LTB 4 to BLT1 expressed on BV fractions
Ligand binding experiments for various BV fractions expressing receptor and G proteins were performed as described under "Experimental Procedures." K d and B max values were derived from the best fit curve to a two-binding-site model using non-linear analysis. ND, not determined.
Sf9 cells infected with His-BLT1 baculovirus released BV on which the BLT1 receptor was expressed. The BV fraction exhibited higher ligand binding activity than the membrane fraction, whereas the membrane fraction contained an excess amount of receptor protein. These results are in good accordance with a previous report that addressed ␤ 2 -adrenergic receptor expression on extracellular virus (23). Human ␤ 2 -adrenergic receptor expressed on the BV of baculovirus exhibited specific ligand binding activity (23). ␤ 2 -Adrenergic receptor is known to couple to the G s class of G protein (24). Because G␣ s is abundantly present in Sf9 cells, the recovered baculovirus contained sufficient intrinsic G s -class G protein such that evidence provided that the functional high affinity receptor was expressed in BV (23).
When BLT1 was expressed alone, the BV fraction exhibited a small number of high affinity and a large number of low affinity ligand binding sites. The K d value of LTB 4 to BLT1 expressed on BV in the high affinity state was 0.126 nM, which is similar to that of human BLT1 expressed in Cos-7 cells previously reported (29). The high affinity and GTP-sensitive binding sites are thought to reflect receptors coupled to G proteins, whereas the low affinity sites represent uncoupled receptors. In Sf9 cells, various G protein subtypes are expressed in different amounts depending on the class of G pro- tein (12,46). The small number of the high affinity binding sites observed in BV expressing BLT1 alone may represent a receptor population coupled to endogenous G proteins.
In this study, we co-infected Sf9 cells with recombinant baculoviruses containing the cDNAs for BLT1 and mammalian heterotrimeric G protein (G␣ i1 ␤ 1 ␥ 2 ). The G␤ 1 ␥ 2 combination used here is capable of interacting with most classes of the G protein ␣ subunit (13,47). In each experiment, the expression of the receptor and G protein subunits was confirmed with immunoblotting (Fig. 3). The expression level of each component in recovered BV correlated well to the amount of recombinant baculovirus used to infect cells. The majority of the receptors expressed in the membrane fraction from Sf9 cells co-infected with BLT1 and G␣ i1 ␤ 1 ␥ 2 recombinant baculoviruses exhibited high affinity ligand binding (Fig. 5, a and c). In the membrane fraction, co-expression of BLT1 and G␣ i1 ␤ 1 ␥ 2 increased high affinity ligand binding sites 3.6-fold compared with BLT1 alone. Even co-expression of BLT1 with G␣ i1 alone led to a 2.8-fold increase in the high affinity binding sites compared with that of BLT1 by itself (Fig. 5c, Table I). Because G␤ subunits have been reported to be detected in Sf9 cells (46), the endogenous G␤␥ subunits of Sf9 cells could well be recruited to the membrane fraction co-expressing receptor and the G␣ subunit and could reconstitute the heterotrimer with exogenous G␣ i1 . The BV released from the co-infected cells also exhibited high affinity for ligand binding comparable with the binding affinity observed in mammalian expression systems (29) (Fig. 5, b and d). For the BV fraction, co-expression of BLT1 and G␣ i1 without G␤␥ resulted in only a slight increase in the high affinity binding sites (Fig. 5, b and d), probably because BV contains a smaller amount of the endogenous G␤␥ subunits. It was necessary for BV to express both G␣ i1 and G␤␥ together with BLT1 to achieve maximum binding (Fig. 5d, Table I). These results are compatible with the finding that G␤␥ complex is required for optimal receptor-G protein interaction (13,17,18,47). The changes in the number of high affinity ligand binding sites on BV depend on the expression of exogenous G protein subunits. From these results, a BV coexpression system could a provide better method for the reconstitution of GPCR and G proteins because the background of the endogenous G proteins is lower than in the cell membrane fraction.
The Sf9 cell expression system has also been used to study the coupling specificity of receptors toward their cognate G proteins (14,17). Interestingly, BLT1 BV co-expressed with G␣ oA ␤ 1 ␥ 2 as well as G␣ i1 ␤ 1 ␥ 2 exhibited high affinity ligand binding (Fig. 8) and also ligand-induced GTP␥S binding (Fig.  9). These data indicate that G␣ oA ␤ 1 ␥ 2 is able to couple to BLT1 and that ligand-induced activation of the receptor led G␣ oA to become a GTP-bound form. Consistent with our findings, a previous report also showed that partially purified LTB 4 receptor from porcine leukocytes was able to couple with exogenous G i -class G proteins, including G␣ o (32). The physiological relevance of the BLT1 signaling pathway mediating G␣ o remains to be elucidated. G␣ o is reported to be expressed only in brain and neuronal tissues (33) and mediates neurotransmitter receptor signaling. Human BLT1 mRNA expresses mainly in peripheral leukocytes and also in spleen and thymus, albeit at low levels (29). Recently, BLT1 expression was reported in C6 astroglioma cells (48). A specific antagonist for BLT1 markedly inhibited ␤-amyloid-induced generation of reactive oxygen in C6 cells (48). BLT1 expression was also detected in the hippocampus of brain and other tissues in a GeneChip analysis of normal human tissues (System Biology and Medicine Data base, LSBM, RCAST, The University of Tokyo, www2.genome.rcast.u-tokyo.ac.jp data base). These observa-tions are suggestive of a physiological function for BLT1 in the central nervous system.
The co-expression of BLT1 with any one of the other G proteins tested (G␣ s , G␣ 11 , G␣ 14 , G␣ 16 , G␣ 12 , and G␣ 13 with G␤ 1 ␥ 2 in every case) had no significant effect on ligand binding (Fig. 8). A previous report showed the G␣ s did not couple to BLT1 in the cAMP response (29). The G␣ 16 , but not G␣ 11 , was considered to be coupled to BLT1 in inositol phosphate production (30). For the BLT1 BV co-expressing heterotrimeric G qclass G protein, we observed a slight difference in the displacement of [ 35 S]GTP␥S binding by GDP in the presence or absence of ligand (Fig. 9, d-f). The rank order of ligand-induced [ 35 S]GTP␥S binding was G␣ 16 Ͼ G␣ 14 Ն G␣ 11 , which was comparable with the previous report (30). For the G 12/13 -class G protein, it is speculated that G␣ 13 also mediates BLT1 signaling functions such as chemotactic attraction of peripheral leukocytes. Although G␣ 12 and G␣ 13 belong to the same class of G proteins, they mediate different signals and generate overlapping effects (5). G␣ 13 has been reported to directly interact with and activate p115 RhoGEF, a guanine nucleotide exchange factor for the GTPase Rho, which activates Rho signaling (49,50). G␣ 13 -mediated Rho signaling regulates a variety of cellular responses such as chemotaxis, cell-cycle progression, and axonal guidance by controlling the organization of the actin cytoskeleton (51). In our findings presented here, BLT1 BV coexpressing G␣ 13 , rather than G␣ 12 , with G␤ 1 ␥ 2 exhibited slightly increased [ 35 S]GTP␥S binding by LTB 4 (Fig. 9, g and  h). Taken together, our data demonstrate that BLT1 expressed on BV conserve the specificity of receptor-G protein coupling seen under physiological conditions. This strongly suggests this expression system should prove highly useful for the combinatorial assessment of coupling specificity of newly identified receptors to various classes of G␣ subunits.
Recently, the chemokine receptors CCR5 and CXCR4 were reported to have incorporated into murine leukemia virus particles. Murine leukemia virus particles containing chemokine receptors were attached to an optical biosensor and were utilized in antibody and human immunodeficiency virus-1 gp120 binding studies (52). Furthermore, it was possible to immobilize the baculovirus BV on a matrix slide with a density of ϳ1000 spots/cm 2 , 2 a technological advance that led to the development of the virus chip. In a baculovirus expression system, the GPCR expressed on BV remain active for at least several months at 4°C (data not shown), except in those rare cases of degradative inactivation. This system is considered to have several advantages over the other available expression systems in terms of application of a biochip sensor. These advantages include the availability of recombinant viruses encoding various G protein subclasses, the reconstitution of high affinity receptors by co-infection of each recombinant virus, as demonstrated here, and the low background of the intrinsic GPCR in Sf9 cells. This system is also expected to be of considerable utility in efforts to characterize the interaction of GPCR and G proteins as well as other molecules, such as the regulator of G protein signaling.