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The human formyl peptide receptor (FPR) is a prototypical Gi protein-coupled receptor, but little is known about quantitative aspects of FPR-Gi protein coupling. To address this issue, we fused the FPR to Giα1, Giα2, and Giα3 and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Gα ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding and GTP hydrolysis and photolabeling of Gα, we demonstrate highly efficient coupling of the FPR to fused Giα1, Giα2, and Giα3without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three Giα isoforms. The Kd values of high affinity agonist binding were ∼100-fold lower than the EC50 (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on theBmax values of agonist saturation binding and ligand-regulated GTPγS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several Gi proteins. Analysis of agonist saturation binding, ligand-regulated GTPγS saturation binding and quantitative immunoblotting with membranes expressing FPR-Giα fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to Giα1, Giα2, and Giα3 with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate Gi proteins linearly and not catalytically.
). GPCRs can isomerize from R to R* spontaneously, and this process is referred to as constitutive activity. Receptor agonists stabilize the R* state and increase basal G protein activity, whereas inverse agonists stabilize the R state and reduce basal G protein activity (
). Based on the comparison ofBmax values of agonist saturation binding and ligand-regulated GTPγS binding, it was proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several Gi proteins (
Several questions regarding the quantitative aspects of FPR-Giα coupling are still unresolved. Specifically, it is unclear why the Kd value for high affinity [3H]fMLF binding is ∼3 nm, whereas the EC50 value of fMLF at activating Gi proteins in terms of GTP hydrolysis, AA-GTP labeling, and GTPγS binding is much higher (∼0.1–1 μm) (
). For Gsα isoforms, it has been shown that the GDP affinity of Gα has a substantial impact on the efficiency of receptor-G protein coupling. Particularly, GsαL (the long splice variant of the α-subunit of the stimulatory G protein of adenylyl cyclase Gs) has a lower GDP affinity than GsαS (the short splice variant) (
). Thus, the GPCR activation energy required for releasing GDP from GsαL is lower than the corresponding activation energy needed for GDP release from GsαS. Accordingly, the β2AR catalyzes GDP release from GsαL more readily than from GsαS. Experimentally, this results in increased efficacy and potency of partial agonists and increased efficacy of inverse agonists when the β2AR is coupled to GsαL as compared with the corresponding ligand properties when coupling of the β2AR to GsαS is considered (
). In other words, GsαL confers to the β2AR the properties of a constitutively active GPCR. Intriguingly, when coupled to Giα2, the FPR is constitutively active as well as assessed by strong inhibitory effects of the inverse agonist CsH on the high basal GTPγS binding (
). Taken together, all these findings raise the question of the impact of the different Giα isoforms on constitutive activity of the FPR.
The aim of our present study was to quantitatively analyze FPR coupling to the three Giα isoforms. To achieve this aim, we fused the FPR to Giα1, Giα2, and Giα3 and expressed the fusion proteins in Sf9 cells. Fusion of GPCR to Gα ensures a defined 1:1 stoichiometry and efficient coupling of the signaling partners and allows for the sensitive detection of differences in the coupling of a given GPCR to different G proteins (
). The baculovirus encoding Giα2 was kindly provided by Dr. A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Recombinant baculovirus encoding the unmodified versions of the G protein subunits β1γ2 was a kind gift of Dr. P. Gierschik (Abteilung für Pharmakologie und Toxikologie, Universität Ulm, Ulm, Germany) (
) was generously provided by Drs. B. Nürnberg and G. Schultz (Institut für Pharmakologie, Freie Universität Berlin, Berlin, Germany). The anti-FLAG Ig (M1 monoclonal antibody) was from Sigma. The antibody recognizing the C terminus of Giα1/2 was from Calbiochem. The anti-His6 Ig was from CLONTECH. [γ-32P]GTP (6000 Ci/mmol), [α-32P]GTP (3000 Ci/mmol), [35S]GTPγS (1100 Ci/mmol), and [3H]fMLF (56 Ci/mmol) were from NEN Life Science Products. [3H]DHA (85–90 Ci/mmol) was from Amersham Pharmacia Biotech. CsH was kindly donated by Novartis. fMLF, fMW, and MLF were from Sigma. Unlabeled nucleotides were obtained from Roche Molecular Biochemicals. All restriction enzymes and T4 DNA ligase were from New England Biolabs. Cloned Pfu DNA polymerase (for generation of fusion protein DNAs) was from Stratagene. Taq polymerase (for analysis of mRNA expression in Sf9 cells) was from Sigma. All other reagents were of the highest purity available and were from standard suppliers.
Construction of the cDNAs Encoding Fusion Proteins
For generation of FPR-Giα fusion proteins, in PCR 1A, the DNA sequence of the C terminus of the FPR was amplified with pGEM3Z-SF-FPR26-His6 as template by using a sense primer 5′ of the newly created EcoRI site of the FPR (
) (senseEcoRI primer) and an antisense primer encoding the hexahistidine tag. The cDNAs of Giα1, Giα2, and Giα3 were amplified in PCR reactions 1B1, 1B2, and 1B3, respectively, using pGEM-2-Giα1,2,3 plasmids as templates. The sense primers annealed with the first 18 bp of the 5′-end of the respective Giα and included the 18 bp of the hexahistidine tag in their 5′-extensions. The antisense primers encoded the 5 C-terminal amino acids of the respective Giα followed by the stop codon and an extra XbaI site for cloning purposes in the 3′-end extension. In PCRs 2A–2C, the cDNA fragments from PCR 1A and PCRs 1B1, 1B2, and 1B3, respectively, were annealed and amplified using the sense EcoRI primer and the antisense primers of PCR 1B1, 1B2, and 1B3, respectively. In this way, a fragment encoding the C terminus of FPR, a hexahistidine tag, and the respective Giα followed by anXbaI site was obtained. The fragments were digested withEcoRI and XbaI and cloned into pGEM3Z-SF-FPR26-His6 digested with EcoRI andXbaI. PCR-generated DNA sequences were confirmed by restriction enzyme analysis and enzymatic sequencing. For cloning into the baculovirus expression vector pVL 1392, the cDNAs encoding fusion proteins in pGEM-3Z were digested with HindIII at the 5′-end of the SF region, blunted with DNA Polymerase I (Klenow fragment), and then digested with XbaI at the 3′-end of Giα. Digested fusion protein DNAs were then transferred into pVL 1392 that had been digested with BglII, blunted with Klenow fragment, and subsequently digested with XbaI. The DNA for the β2AR-Giα2fusion protein (used as immunoblotting standard for the determination of Giα2 expression) was prepared by overlap extension PCR analogous to the FPR-Giα fusion protein DNAs. PCR-generated sequences were confirmed by enzymatic sequencing.
Generation of Recombinant Baculoviruses, Cell Culture, and Membrane Preparation
Generation of baculoviruses, cell culture, and membrane preparation were performed exactly as described (
). Sf9 cells were co-infected with recombinant baculoviruses encoding nonfused FPR, Giα2, and G protein β1γ2 complex or fusion proteins plus G protein β1γ2 complex.
Analysis of FPR-Giα Fusion Proteins by Agonist Saturation Binding, GTPγS Binding, and GTPase Activity
[3H]fMLF saturation binding, GTPγS saturation binding, and time course of GTPγS binding in Sf9 membranes expressing FPR-Giα fusion proteins plus β1γ2 complex were performed exactly as described for Sf9 membranes expressing nonfused FPR, Giα2, and β1γ2complex (
). Steady-state GTPase activity with different substrate concentrations was determined as described for Sf9 membranes expressing β2AR-GsαL fusion protein except that the MgCl2 concentration was 5 mm instead of 1 mm (
). Briefly, Sf9 membranes expressing FPR-Giα fusion proteins (200 μg/tube) were incubated for 20 min at 25 °C in a buffer containing 100 mm NaCl, 5 mm MgCl2, 1 μm GDP, 5 μg/ml soybean trypsin inhibitor, and 30 mm HEPES/NaOH, pH 7.5. Reaction mixtures additionally contained 1 μCi of AA-GTP and solvent (control) or 10 μm fMLF. After centrifugation for 10 min at 15,000 × g and 4 °C, membranes were suspended in a buffer containing 100 mm NaCl, 5 mmMgCl2, 5 μg/ml soybean trypsin inhibitor, 2 mm dithiothreitol, and 30 mm HEPES/NaOH, pH 7.5. Membranes were UV-irradiated for 3 min at 302 nm and separated by SDS-PAGE, and AA-GTP-labeled proteins were identified by autoradiography.
Analysis of FPR-Giα Fusion Protein Expression on the Protein Level
Sf9 membranes were analyzed by immunoblotting using the monoclonal M1 antibody, which recognizes the N-terminal FLAG epitope of the FPR and β2AR (
), anti-Giα1/2 Ig, and anti-Giα3 Ig. The M1 antibody was used at a dilution of 1:1000. The other antibodies were used at a dilution of 1:500. SDS-PAGE and immunoblotting were performed exactly as described (
Analysis of FPR-Giα Fusion Protein Expression on the mRNA Level
mRNA from Sf9 cells infected with recombinant baculoviruses was isolated with the RNeasy kit from Qiagen and treated with RNase-free DNase. mRNA was reverse-transcribed using the First Strand cDNA synthesis kit from Amersham Pharmacia Biotech. The cDNAs of Giα1 and Giα2 in FPR-Giα fusion proteins were amplified using appropriate primer pairs. PCR products were digested with various restriction enzymes, separated by electrophoresis on gels containing 2% (w/v) agarose, and visualized by ethidium bromide staining.
Protein was determined using the Bio-Rad DC protein assay kit. Data were analyzed by nonlinear regression, using the Prism program.
Analysis of FPR-Giα Fusion Protein Expression on the Protein and mRNA Level
The human FPR expressed in Sf9 cells has a molecular mass of 40 kDa (
) detected antigens of the appropriate mass in membranes prepared from baculovirus-infected Sf9 cells (Fig. 1A). The anti-Giα1/2 Ig detected the FPR-Giα1 and FPR-Giα2 fusion proteins but not FPR-Giα3 (Fig. 1B). In contrast, the anti-Giα3 Ig reacted with the FPR-Giα3 fusion protein but not with FPR-Giα1 or FPR-Giα2 (Fig. 1C). There was no indication for proteolytic degradation of fusion proteins. Because the anti-Giα1/2 Ig cannot discriminate between Giα1 and Giα2 (Fig.1B), we performed reverse transcriptase-PCR analysis on mRNA from Sf9 cells infected with the FPR-Giα1 and FPR-Giα2 baculoviruses to differentiate between the two Giα isoforms. Giα1 has a unique SacI site at position 382, and Giα2 has a uniqueBamHI site at position 653 (Fig. 1D). The Giα portions of fusion proteins were amplified by PCR and digested. SacI generated the expected 382- and 711-bp fragments with Giα1, whereas Giα2 cDNA was not cut. In contrast, digestion with BamHI yielded the expected 443- and 653-bp fragments with Giα2, whereas Giα1 cDNA was not cut. Taken together, the immunoblotting and reverse transcriptase-PCR data document the specific expression of FPR-Giα1, FPR-Giα2, and FPR-Giα3 fusion proteins in Sf9 cell membranes.
[3H]fMLF Binding Studies
Fig.2 (A and B) shows representative [3H]fMLF saturation binding curves for FPR-Giα1, and TableI summarizes the data for all three fusion proteins. For comparison, the [3H]fMLF binding data for nonfused FPR co-expressed with Giα2are included as well in Table I. FPR-Giα fusion proteins bound the agonist [3H]fMLF (0.2–30 nm) according to single-site saturation curves. The Kd and Bmax values of [3H]fMLF binding to FPR-Giα1, FPR-Giα2, and FPR-Giα3 were similar to each other and comparable with the values for the co-expression system. An increase of the [3H]fMLF concentration up to 300 nm in the fusion protein and co-expression systems did not further increaseBmax of agonist binding as compared with a [3H]fMLF concentration of 30 nm (data not shown), indicating that a possible low affinity agonist-binding site of the FPR cannot be unmasked by means of agonist saturation binding. Binding of GTPγS to Gα uncouples GPCRs from G proteins and reduces high affinity agonist binding (
). By analogy to nonfused FPR co-expressed with Giα2, GTPγS substantially reduced the Bmax of [3H]fMLF binding to FPR-Giα fusion proteins and, to a variable extent, increased the Kd for [3H]fMLF.
Table I[ 3 H]fMLF saturation binding in Sf9 membranes expressing FPR-G i α fusion proteins: comparison with nonfused FPR co-expressed with G i α 2
FPR + Giα2
4.51 ± 1.08
4.62 ± 1.11
4.54 ± 0.73
3.41 ± 0.82
1.20 ± 0.28
0.90 ± 0.13
1.28 ± 0.20
1.18 ± 0.09
6.96 ± 1.82
24.91 ± 6.62
14.16 ± 3.12
13.24 ± 4.48
Bmax GTPγS (pmol/mg)
0.19 ± 0.02
0.32 ± 0.05
0.45 ± 0.05
0.34 ± 0.06
Membranes from Sf9 membranes expressing FPR-Giα fusion proteins were incubated in the presence of [3H]fMLF at concentrations of 0.2–30 nm in the absence and presence of 10 μm GTPγS. Nonspecific binding, i.e. the [3H]fMLF binding not competed for by 10 μmunlabeled fMLF, was subtracted from both sets of binding data. Data were analyzed for best fit to single-site and two-site saturation curves. All curves were best fit to single-site saturation curves. Data shown are the means ± S.D. of three experiments with different membrane preparations performed in triplicates. The data for the nonfused FPR co-expressed with Giα2 were taken from Ref.
Ligand-regulated GTPγS binding to G proteins is a sensitive method for studying GPCR-G protein coupling, specifically for analyzing the time course of G protein activation and the stoichiometry of coupling (
). Fig.2C shows a representative time course experiment for the GTPγS binding to FPR-Giα1, and Fig.2D shows a representative GTPγS saturation binding experiment for FPR-Giα1. TableII summarizes the GTPγS binding data for all three fusion proteins. For comparison, the GTPγS binding data for the FPR co-expressed with Giα2 are included in Table II as well. As reported previously for the FPR co-expressed with Giα2 in Sf9 cells (
), the FPR fused to Giα2 displayed high constitutive activity as assessed by the strong inhibitory effect of the inverse agonist CsH on GTPγS binding. CsH also displayed strong inhibitory effects at the FPR fused to Giα1and Giα3. The Kd values for fMLF-stimulated and CsH-inhibited GTPγS binding were similar for all FPR-Giα fusion proteins studied and compare favorably with the Kd values for GTPγS binding with nonfused FPR co-expressed with Giα2. With respect to the time course of GTPγS binding, fMLF reduced whereas CsH increasedt
, but there was some variability between the different systems studied.
Table IIEffects of fMLF and CsH on [ 35 S]GTPγS binding in Sf9 membranes expressing FPR-G i α fusion proteins and nonfused FPR with G i α 2 : saturation binding and time course
FPR + Giα2
1.39 ± 1.00
0.91 ± 0.60
0.66 ± 0.34
0.78 ± 0.17
Bmax fMLF (pmol/mg)
3.08 ± 0.39
3.24 ± 0.12
1.35 ± 0.22
2.83 ± 0.18
1.10 ± 0.56
1.15 ± 0.32
0.82 ± 0.82
1.84 ± 0.67
Bmax CsH (pmol/mg)
2.57 ± 0.67
2.08 ± 0.32
1.75 ± 0.06
3.03 ± 0.22
Bmax total (pmol/mg)
5.65 ± 1.07
5.33 ± 0.20
3.11 ± 0.15
5.86 ± 0.19
11.2 ± 7.3
9.4 ± 4.0
14.4 ± 6.2
17.0 ± 3.5
8.2 ± 5.2
5.2 ± 3.1
6.9 ± 1.1
5.1 ± 2.3
22.3 ± 5.2
15.6 ± 2.3
22.7 ± 12.0
35.9 ± 6.9
Membranes from Sf9 cells expressing FPR-Giα fusion proteins were prepared. For saturation binding experiments, membranes were incubated for 60 min in the presence of 0.1–1 nm[35S]GTPγS plus unlabeled GTPγS at different concentrations to give final ligand concentrations of 0.1–10 nm, 1 μm GDP, and solvent (basal), fMLF (10 μm), or CsH (10 μm). Nonspecific binding was determined in the presence of 10 μm unlabeled GTPγS. For each GTPγS concentration, the basal GTPγS binding value was subtracted from the GTPγS binding value observed in the presence of fMLF to calculate the increase in GTPγS binding caused by fMLF. From each basal GTPγS binding value, the GTPγS binding value observed in the presence of CsH was subtracted to obtain the decrease of GTPγS binding caused by CsH. Saturation binding data were analyzed for best fit to single-site and two-site saturation curves. All data were best fit to single-site saturation curves. For time course experiments, Sf9 membranes were incubated for 5–180 min in the presence of 1 nm [35S]GTPγS plus 9 nm unlabeled GTPγS, 1 μm GDP, and solvent (basal), fMLF (10 μm), or CsH (10 μm). Time course data were analyzed for best fit to monophasic and biphasic saturation curves. All data best fit to a monophasic saturation curve. Data shown are the means ± S.D. of three experiments with different membrane preparations performed in triplicates. The data for the nonfused FPR co-expressed with Giα2 were taken from Ref.
), 1 mol of activated GPCR can maximally stimulate the binding of 1 mol of GTPγS to its fused Gα partner. Thus, one would expect that the Bmax of receptor ligand binding is very similar to the Bmax of ligand-regulated GTPγS binding. The Bmax of ligand-regulated GTPγS binding (Bmax total) is the difference between maximum GTPγS binding stimulated by fMLF and minimum GTPγS binding inhibited by CsH. As is evident from the comparison of Bmax values for [3H]fMLF binding and Bmax totalvalues of GTPγS binding (compare Tables I and II and Fig. 2,A, B, and D),Bmax total of GTPγS binding surpassesBmax of [3H]fMLF binding by 4.7-fold for FPR-Giα1, by 5.9-fold for FPR-Giα2, and by 2.5-fold for FPR-Giα3.
Resolution of the Discrepancies between the Bmax of [3H]fMLF Binding and Ligand-regulated GTPγS Binding
Two explanations for the discrepancies between theBmax values of [3H]fMLF binding and ligand-regulated GTPγS binding have to be considered. First, the fused FPR could interact not only with its fused Giα partner but also with G proteins of the host cell. Indeed, cross-talk of a fused Gi protein-coupled GPCR to endogenous G proteins occurs in some mammalian expression systems (
). Second, the FPR could still couple only to its fused Gα partner but may do so in a state of low agonist affinity. This low agonist affinity state may not be detected in the [3H]fMLF binding assay but in the GTPγS binding assay because the receptor promotes binding of GTPγS to the G protein. The efficient coupling of GPCRs to G proteins in a state of low agonist affinity had already been postulated in previous studies (
). These studies were performed in the presence of NaCl (see “Experimental Procedures”) to facilitate detection of agonist responses and to suppress agonist-independent labeling of G proteins as the result of high constitutive receptor activity (
). fMLF efficiently stimulated the incorporation of AA-GTP into all three FPR-Giα fusion proteins (Fig. 3). However, fMLF did not stimulate the incorporation of AA-GTP into ∼40–45-kDa proteins,i.e. endogenous insect cell G proteins (
). These data rule out the possibility of cross-talk of the fused FPR to endogenous G proteins of Sf9 cells. In agreement with the data regarding fused FPR, nonfused FPR also does not couple to insect cell G proteins (
To address the second explanation, we determined the expression of FPR-Giα fusion proteins by a method that is independent of radioligand binding. Specifically, we took advantage of the FLAG and His6 epitopes that are located at the N and C termini, respectively, of the GPCR portions of fusion proteins (see “Experimental Procedures”) (
). The previously characterized β2AR-GsαL fusion protein can be used as standard to assess the expression level of other proteins bearing the same epitopes because the expression level of β2AR-GsαL can be unequivocally determined by receptor antagonist saturation binding (
). Fig.4A shows that the immunoreactivity of FPR-Giα1 expressed at 1.4 pmol/mg ([3H]fMLF saturation binding) was even higher than the immunoreactivity of β2AR-GsαL expressed at 8.6 pmol/mg ([3H]DHA saturation binding) using the anti-His6 Ig. The signals with FPR-Giα2 (Fig. 4A) and FPR-Giα3 (data not shown) expressed at 1.0 pmol/mg each were slightly smaller than the signals obtained with β2AR-GsαL expressed at 8.6 pmol/mg. By using the anti-FLAG Ig, the immunoreactivity with β2AR-GsαL expressed at 8.6 pmol/mg was moderately higher than with FPR-Giα1 expressed at 1.4 pmol/mg. The difference in sensitivity between the anti-FLAG Ig and anti-His6 Ig could be due to the fact that the FPR is heavily glycosylated at the extreme N terminus (
) and that this glycosylation could interfere with the recognition of the N-terminal FLAG epitope by the M1 antibody.
To answer the question of whether [3H]fMLF saturation binding also underestimates the expression level of nonfused FPR, we compared the immunoreactivity of membranes expressing the β2AR or the FPR plus Giα2 plus β1γ2 complex using the anti-FLAG Ig. Fig.4C shows that the FPR runs as a much more diffuse band in SDS-PAGE than the β2AR, indicating that the FPR is more heavily glycosylated than the β2AR. Despite this difference in glycosylation pattern of the two GPCRs, it is evident that the immunoreactivity of the β2AR expressed at 3.9 pmol/mg ([3H]DHA saturation binding) is comparable with the immunoreactivity of the FPR expressed at 1.1 pmol/mg ([3H]fMLF saturation binding). Unfortunately, we could not visualize nonfused GPCRs with the His6 Ig. Apparently, the His6 Ig cannot efficiently recognize the His6 tag when directly located at the C terminus of the antigens studied.
Taken together, the quantitative immunoblotting studies demonstrate that [3H]fMLF saturation binding underestimates the actual expression level of the fused and nonfused FPR by a factor of ∼4–8, depending on which antibody is used for the detection of the GPCRs. This factor agrees well with the factors obtained in the [3H]fMLF and GTPγS saturation binding studies (Tables I and II). The similarity of the fusion protein expression levels as determined by immunoblotting and GTPγS saturation binding also implies that the majority, if not all, of the expressed fusion protein molecules are functionally active. Of interest, by comparing theBmax values of agonist and antagonist binding at the adenosine A1 receptor co-expressed with, or fused to, Giα proteins, it was also shown that agonist binding underestimates GPCR expression level by ∼3–6-fold (
). Unfortunately, an antagonist radioligand for the FPR is not available, but the combination of immunoblotting and ligand-regulated GTPγS binding can compensate for this deficiency.
We also assessed the potency of fMLF at activating the fused Giα partner. If the FPR in a state of low agonist affinity does indeed couple to Gi proteins, then the EC50 values for fMLF in functional assays should be considerably higher than the Kd values in the agonist binding studies. In fact, at all three fusion proteins, fMLP activated GTP hydrolysis with EC50 values that are ∼80–125-fold higher than the Kd values for high affinity [3H]fMLF binding (compare Fig. 2 (A,B, and E) and Tables I andIII). A similar discrepancy between agonist affinity and agonist potency was reported for nonfused FPR expressed in HL-60 leukemia cells and Sf9 cells (
Table IIISteady-state GTPase activity in Sf9 membranes expressing FPR-G i α fusion proteins: efficacy and potency of FPR ligands
0.56 ± 0.25
0.36 ± 0.27
0.49 ± 0.25
0.66 ± 0.14
8.46 ± 1.16
0.77 ± 0.05
8.90 ± 4.23
0.86 ± 0.03
4.73 ± 2.41
0.44 ± 0.07
9.35 ± 3.24
0.45 ± 0.08
4.97 ± 2.44
0.53 ± 0.09
2.21 ± 1.09
−0.50 ± 0.05
0.30 ± 0.27
−0.65 ± 0.11
1.02 ± 0.35
−0.56 ± 0.23
0.39 ± 0.18
−0.01 ± 0.01
−0.07 ± 0.03
−0.03 ± 0.02
Membranes expressing FPR-Giα fusion proteins were incubated in the presence of 100 nm [γ-32P]GTP (0.3 μCi/tube), solvent (2% v/v Me2SO) or ligands at the following concentrations; fMLF (1 nm to 100 μm), fMW (1 nm to 1 mm), MLF (1 nm to 20 μm), CsH (1 nm to 20 μm), BocFLFLF (1 nm to 20 μm). Concentration-response curves were best fit to sigmoidal saturation curves. EC50 values for agonists and IC50 values for CsH, corresponding to ligand potencies, were obtained from fitted sigmoidal saturation curves. The efficacies of ligands were calculated by dividing the maximal ligand-stimulated (or CsH-inhibited) GTPase activity by the maximal fMLF-stimulated GTPase activity. The maximum effect of fMLF was set 1.00. The maximum ligand effects were derived from sigmoidal saturation curves, too. Data shown are the means ± S.D. of three experiments with different membrane preparations performed in triplicates. NA, not applicable (because of the minimal effects of BocFLFLF, precise potencies could not be calculated).
). However, because agonist saturation binding largely underestimates the actual expression level of FPR-Giα fusion proteins (Tables I and II and Figs. 2, A,B, and D, and 4), we divided GTPase activities by the Bmax values of ligand-regulated GTPγS binding. Fig. 2F shows a representative experiment for the kinetics of ligand-regulated GTP turnover at FPR-Giα1, and TableIV summarizes the data for all three fusion proteins. The Km values of fMLF-stimulated GTP hydrolysis for FPR-Giα fusion proteins were similar to each other as were the Km values for CsH-inhibited GTP hydrolysis. The Vmax values of ligand-regulated GTP turnover were similar for the three FPR-Giα fusion proteins. The kinetic parameters of the GTPase of FPR-Giα fusion proteins are also similar to the corresponding parameters of an α2-adrenoreceptor-Giα1 fusion protein (
Table IVKinetics of steady-state GTP hydrolysis in Sf9 membranes expressing FPR-G i α fusion proteins
0.16 ± 0.03
0.10 ± 0.05
0.18 ± 0.03
1.14 ± 0.45
0.55 ± 0.11
0.78 ± 0.35
0.10 ± 0.00
0.02 ± 0.03
0.07 ± 0.06
Vmax CsH (min−1)
0.37 ± 0.12
0.26 ± 0.05
0.27 ± 0.17
Vmax total (min−1)
1.51 ± 0.57
0.81 ± 0.16
1.05 ± 0.52
Membranes expressing FPR-Giα fusion proteins were incubated in the presence of 30 nm to 1.5 μm[γ-32P]GTP (0.5 μCi/tube) and solvent (basal), fMLF (10 μm), or CsH (10 μm). For each GTP concentration, the basal GTPase activity was subtracted from the GTPase activity observed in the presence of fMLF to calculate the increase in GTPase activity caused by fMLF. From each basal GTPase activity value, the GTPase activity observed in the presence of CsH was subtracted to obtain the decrease in GTPase activity caused by CsH. Kinetic data were analyzed for best fit to single-site and two-site saturation curves. All data fit best to single-site saturation curves. To obtain GTP turnover numbers, GTPase activities (expressed as pmol/mg/min) were divided by the Bmax total (pmol/mg) of ligand-regulated GTPγS binding of the respective membrane (see TableII) because this is a more precise measure of fusion protein expression level than receptor agonist binding (see Table I and Fig. 4).Vmax total is the sum of fMLF- and CsH-regulated GTP turnover (ligand-regulated GTP turnover). Data shown are the means ± S.D. of three experiments with different membrane preparations performed in triplicates.
The data obtained with the full agonist fMLF and the full inverse agonist CsH in GTPγS binding and GTPase studies (Tables Table II, Table III, Table IV) point to high constitutive activity of the FPR coupled to all Giα isoforms. We wished to explore the hypothesis that there are, nonetheless, subtle differences in the constitutive activity of the FPR coupled to Giα isoforms that can only be detected with partial agonists. According to the extended ternary complex model, an increase in constitutive activity is accompanied by an increase in partial agonist efficacy and potency (
We analyzed a panel of peptides and identified fMW as moderately strong partial agonist and the nonformylated peptide MLF as weak partial agonist at the FPR. Fig. 2E shows representative concentration-response curves for various FPR ligands at FPR-Giα1, and Table III provides a summary for all three FPR-Giα fusion proteins studied. In all systems studied, FPR ligands activated GTP hydrolysis in the following order of efficacy: fMLF > fMW > MLF > BocFLFLF (ineffective). There were no systematic differences in agonist efficacy and potency for a given fusion protein compared with another fusion protein. Specifically, fMW and MLF were not more efficacious and potent at FPR-Giα2 than at FPR-Giα1 and FPR-Giα3.
Expression Level of Nonfused Giα2 in the Co-expression System
When the FPR-Giα2fusion protein was compared with the corresponding co-expression system, we were puzzled that not only the Bmaxvalues of [3H]fMLF binding were similar to each other but also the Bmax values of ligand-regulated GTPγS binding (compare Tables I and II). However, if the FPR had activated Giα catalytically in the co-expression system, we would have expected a much higher Bmax of ligand-regulated GTPγS binding in this system than in the fusion protein system. Thus, we had to address the hypothesis that the lowBmax of ligand-regulated GTPγS binding in the co-expression system was due to a low expression level of Gi proteins, resulting in poor availability of G proteins for activation by GPCR. We used a β2AR-Giα2 fusion protein expressed at 15 pmol/mg ([3H]DHA saturation binding) as standard for analyzing the expression level of nonfused Giα2. The immunoreactivity with the anti-Giα1/2 Ig in membranes expressing FPR plus Giα2 was ∼20–30 times higher than the immunoreactivity with similar amounts of protein from membranes expressing β2AR-Giα2. Thus, in the co-expression system, Giα2 is expressed at a level of ∼300–450 pmol/mg. Similar expression levels in Sf9 cells were also reported for other G proteins (
). Given an estimated FPR expression level of ∼4 pmol/mg in the co-expression system (Fig. 4C), these data clearly show that limited expression of Giα2 cannot account for the surprisingly low Bmax of ligand-regulated GTPγS binding in this system.
Highly Efficient Receptor-G Protein Coupling in a FPR-Giα2 Fusion Protein
The aim of our study was to quantitatively analyze the coupling of the FPR to the three Giα isoforms. The fusion protein technique provides a defined 1:1 stoichiometry of the signaling partners and is a sensitive system for dissecting differences in the coupling of a given GPCR to different Gα isoforms (
). However, because the fusion of a receptor C terminus to the Gα N terminus is artificial, one may be concerned that the fusion substantially alters the properties of the receptor and the G protein as well as their coupling to each other. Moreover, in certain expression systems, Gi protein-coupled receptors cannot only couple to their fused Giα partner but also to the endogenous G proteins of the host cells (
). To address these concerns, we compared the coupling of the FPR to Giα2 in the fused and nonfused state and visualized activated G proteins by photoaffinity labeling. There were only minor differences in FPR-Giα2 coupling between the fused and nonfused state (Tables I and II), and there is no cross-talk between the fused FPR and G proteins of the host cell (Fig.4). Thus, the fusion protein technique is a valid approach to analyze potential differences in the coupling of the FPR to Giα isoforms.
In the co-expression system, there is an ∼100-fold molar excess of Giα2 over FPR as assessed by quantitative immunoblotting with defined standards (Figs. 4C and5). Despite the large excess of G protein relative to receptor in the co-expression system and the high absolute expression levels of FPR and Giα2, the signaling efficiency of the co-expression system as assessed by theBmax of ligand-regulated GTPγS binding was not greater than that of the fusion protein system, which has only a 1:1 stoichiometry of receptor to G protein and a much lower G protein expression level (Table II). These data show that in the co-expression system, the vast majority of G proteins is not engaged in coupling, whereas in the fusion protein system, most if not all G proteins participate in coupling. Thus, the fusion induces an optimal positioning of Giα relative to the FPR. Similar conclusions were obtained for β2AR-Gsα and adenosine A1 receptor-Giα fusion proteins (
Similarly Efficient Coupling of the FPR to Giα1, Giα2, and Giα3
A stimulus for our present study was observations regarding the importance of the GDP affinity of G proteins for receptor-G protein coupling. Specifically, the GDP affinity of Giα2 is lower than the GDP affinity of Giα1 and Giα3 (
). An explanation for these findings could be that the lower the GDP affinity of a given Gα is, the lower is the GPCR activation energy required to catalyze GDP release from a G protein. Because even an agonist-free GPCR can efficiently promote GDP release from a G protein with low GDP affinity, this G protein accordingly confers to the coupled GPCR the properties of constitutive activity.
Based on the above findings and considerations, we predicted that the FPR coupled to Giα2 would have a higher degree of constitutive activity than the FPR coupled to Giα1 and Giα3. Surprisingly, however, the relative inhibitory effects of CsH at the total ligand-regulated GTPγS binding and GTP hydrolysis were very similar among the three Giα isoforms (Tables Table II, Table III, Table IV). Another indicator for increased constitutive activity of a GPCR is elevated efficacy and potency of partial agonists (
). However, we could not detect consistent increases in these parameters for FPR-Giα2 as compared with FPR-Giα1 and FPR-Giα3 (Table IV). Taken together, our data show that the different Giα isoforms, which differ from each other in GDP affinity, do not have a specific impact on the constitutive activity of the FPR. These data clearly demonstrate that the observations made for the coupling of Gsα splice variants to the β2AR cannot be readily extrapolated to other GPCR-G protein pairs. Possibly, the intrinsic constitutive activity of the FPR is so high that the modulation of constitutive activity by GDP affinity of Giα isoforms is too subtle to become effective.
The lack of substantial differences in the coupling of the FPR to Giα isoforms is somewhat unexpected in view of the fact that many GPCRs show differences in coupling efficiency to Giα isoforms, that Giα2 is the major G protein in phagocytes, and that Giα1is not expressed in these cells (
). Thus, the extensive contact area of the FPR with G proteins may provide the structural basis for the lack of differential coupling to Giα isoforms. Based on our findings, one can assume that in vivo, Giα2 and Giα3 are used interchangeably by the FPR.
Stoichiometry of FPR-Giα Coupling: Implications for Signaling in Vivo
Because FPR-Giα fusion proteins have a defined 1:1 stoichiometry of GPCR and Gα and because there is no cross-talk of the fused receptor with endogenous G proteins, we expected very similar Bmax values for [3H]fMLF binding and ligand-regulated GTPγS binding, reflecting the fact that one FPR molecule can activate one Giα molecule. However, the Bmax total values of GTPγS binding were up to 6-fold higher than the Bmax values of [3H]fMLF binding (Tables I and II). Thus, a substantial fraction of the FPRs appears to exist in a state of low agonist affinity that cannot be detected in the agonist binding assay but which, nonetheless, efficiently couples to G proteins to stimulate guanine nucleotide exchange. This model is supported by the fact that the EC50values of fMLF at activating GTP hydrolysis and guanine nucleotide binding to Giα in fused and nonfused systems are ∼100-fold higher than the Kd values for [3H]fMLF binding (
). Additionally, quantitative immunoblotting with anti-His6 Ig and anti-FLAG Ig using β2AR-GsαL as standard demonstrated that agonist saturation binding greatly underestimates the actual expression level of FPR fusion proteins (Fig. 4, Aand B). The underestimation of FPR expression level by [3H]fMLF binding is also true for nonfused receptors (Fig. 4C).
The fact that the agonist binding assay underestimates the actual expression level of FPRs by a factors of ∼2.5–6 renders it necessary to re-evaluate previously obtained conclusions regarding the stoichiometries of FPR-Gi protein coupling in nonfused systems. We and other groups had proposed that the FPR activates Gi proteins catalytically, i.e. a single FPR molecule activates several Giα molecules (
). These numbers were obtained by dividing Bmax of ligand-regulated GTPγS binding by Bmax of [3H]fMLF binding. If one uses the actualBmax of FPR expression for these calculations, it emerges that a single FPR in average does not activate much more than a single Gi protein, i.e. there is linear rather than catalytical G protein activation. This model of linear signal transfer is further supported by the fact that in the co-expression system, despite very high abundance of Giproteins, the total number of activated Gi proteins was not higher than in the fusion protein system (Tables I and II and Fig. 5). In a co-expression system of the β2AR and GsαL we also could not obtain evidence for catalytical G protein activation by receptors (
). Collectively, our data suggest that the 1: 1 stoichiometry of GPCR and Gα obtained in fusion proteins may reflect the in vivo stoichiometry of receptor-G protein coupling more closely than was previously appreciated (
Linear G protein activation does have to result in a loss of signal amplification and stimulus sensitivity. Given the fact that the FPR can couple to Gi proteins even in a state of low agonist affinity (Tables Table I, Table II, Table III and Fig. 2E) (
), our model of linear signal transfer could explain the different phagocyte responses over a large range of fMLF concentrations. Specifically, at low fMLF concentrations, phagocytes migrate along a fMLF gradient toward their target, i.e. the formyl peptide-releasing bacteria (
). Once the phagocyte is approaching the bacteria, the concentration of formyl peptide increases, and more G proteins are activated, resulting in stimulation of cytotoxic cell functions such as lysosomal enzyme release and superoxide radical production and destruction of bacteria (
). Based on our present data, it is likely that the actual FPR expression level in phagocytes is considerably higher than 1 pmol/mg. Thus, the FPR is presumably a GPCR with one of the highest physiological expression levels.
In conclusion we have shown that the human FPR couples to the Gi proteins Giα1, Giα2, and Giα3 with similar efficiency. By taking advantage of the defined 1:1 stoichiometry of GPCR and Gα in fusion proteins, we obtained insights into quantitative aspects of FPR-Gi protein coupling that could not have been achieved by another approach. Our data suggest that the FPR activates Gi proteins in a linear fashion and not catalytically.
We thank Dr. Brian K. Kobilka for helpful discussions and the reviewers of this paper for most valuable critique and suggestions.