J Biol Chem, Vol. 274, Issue 47, 33259-33266, November 19, 1999

Quantitative Analysis of Formyl Peptide Receptor Coupling to G_i1, G_i2, and G_i3^*

Katharina Wenzel-Seifert^§¶, John M. Arthur, Hui-Yu Liu^§, and Roland Seifert^§

From the  Higuchi Biosciences Center and the ^§ Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas 66045-2505 and the  Department of Medicine, Kidney Disease Program, University of Louisville, Louisville, Kentucky 40202

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human formyl peptide receptor (FPR) is a prototypical G_i protein-coupled receptor, but little is known about quantitative aspects of FPR-G_i protein coupling. To address this issue, we fused the FPR to G_i1, G_i2, and G_i3 and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to G 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) (GTPS) binding and GTP hydrolysis and photolabeling of G, we demonstrate highly efficient coupling of the FPR to fused G_i1, G_i2, and G_i3 without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G_i isoforms. The K_d 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 the B_max values of agonist saturation binding and ligand-regulated GTPS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G_i proteins. Analysis of agonist saturation binding, ligand-regulated GTPS saturation binding and quantitative immunoblotting with membranes expressing FPR-G_i 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 G_i1, G_i2, and G_i3 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 G_i proteins linearly and not catalytically.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most intercellular signal molecules exert their effects through GPCRs¹ that couple to heterotrimeric G proteins, which in turn regulate the activity of effector systems (1-3). The extended ternary complex model of receptor activation assumes that GPCRs exist either in an inactive "R" state or an active "R*" state (4-6). 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 (4, 6, 7).

The FPR is expressed predominantly in phagocytic cells and plays a crucial role in host defense against bacterial infections (8-10). The FPR couples to the pertussis toxin-sensitive G proteins G_i2 and G_i3 (11-13) and, via released G protein -subunits, mediates activation of phospholipase C-2 (14, 15) and phosphatidylinositol-3-kinase (16-18). Consequently, phagocyte functions such as chemotaxis, lysosomal enzyme release, and superoxide radical formation are activated (8, 19-21). Based on the comparison of B_max values of agonist saturation binding and ligand-regulated GTPS binding, it was proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G_i proteins (22-25).

Several questions regarding the quantitative aspects of FPR-G_i coupling are still unresolved. Specifically, it is unclear why the K_d value for high affinity [³H]fMLF binding is ~3 nM, whereas the EC₅₀ value of fMLF at activating G_i proteins in terms of GTP hydrolysis, AA-GTP labeling, and GTPS binding is much higher (~0.1-1 µM) (8, 12, 25, 26). Similarly, it is unknown why fMLF is much more potent at activating chemotaxis than lysosomal enzyme release and superoxide radical formation (8, 19-21). Furthermore, it is not clear whether the FPR couples differentially to G_i isoforms.

This latter question is intriguing in view of several findings. First, phagocytes express G_i2 at a much higher level than G_i3, and G_i1 is not expressed at all (11, 13, 27, 28). Second, certain GPCRs, including the receptors for the chemoattractant interleukin-8, differ substantially in their coupling efficiency to various G_i isoforms (29-34). Third, data obtained with the G_i2 knock-out mouse suggest that this G protein has unique functions in signal transduction (35, 36).

Perhaps most importantly, G_i2 has a lower GDP affinity than G_i1 and G_i3 (37, 38). For G_s isoforms, it has been shown that the GDP affinity of G has a substantial impact on the efficiency of receptor-G protein coupling. Particularly, G_sL (the long splice variant of the -subunit of the stimulatory G protein of adenylyl cyclase G_s) has a lower GDP affinity than G_sS (the short splice variant) (7, 39). Thus, the GPCR activation energy required for releasing GDP from G_sL is lower than the corresponding activation energy needed for GDP release from G_sS. Accordingly, the ₂AR catalyzes GDP release from G_sL more readily than from G_sS. Experimentally, this results in increased efficacy and potency of partial agonists and increased efficacy of inverse agonists when the ₂AR is coupled to G_sL as compared with the corresponding ligand properties when coupling of the ₂AR to G_sS is considered (7). In other words, G_sL confers to the ₂AR the properties of a constitutively active GPCR. Intriguingly, when coupled to G_i2, the FPR is constitutively active as well as assessed by strong inhibitory effects of the inverse agonist CsH on the high basal GTPS binding (25). Taken together, all these findings raise the question of the impact of the different G_i isoforms on constitutive activity of the FPR.

The aim of our present study was to quantitatively analyze FPR coupling to the three G_i isoforms. To achieve this aim, we fused the FPR to G_i1, G_i2, and G_i3 and expressed the fusion proteins in Sf9 cells. Fusion of GPCR to G 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 (7, 40-43).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The cDNAs of G_i1, G_i2 and G_i3 in pGEM-2 were kindly provided by Dr. R. Reed (Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD) (44). The baculovirus encoding G_i2 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 ₁₂ was a kind gift of Dr. P. Gierschik (Abteilung für Pharmakologie und Toxikologie, Universität Ulm, Ulm, Germany) (45). The antibody recognizing the C terminus of G_i3 (AS 86) (46) 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 G_i1/2 was from Calbiochem. The anti-His₆ Ig was from CLONTECH. [-³²P]GTP (6000 Ci/mmol), [-³²P]GTP (3000 Ci/mmol), [³⁵S]GTPS (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-G_i 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 (25) (sense EcoRI primer) and an antisense primer encoding the hexahistidine tag. The cDNAs of G_i1, G_i2, and G_i3 were amplified in PCR reactions 1B₁, 1B₂, and 1B₃, respectively, using pGEM-2-G_i1,2,3 plasmids as templates. The sense primers annealed with the first 18 bp of the 5'-end of the respective G_i 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 G_i 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 G_i followed by an XbaI site was obtained. The fragments were digested with EcoRI and XbaI and cloned into pGEM3Z-SF-FPR26-His₆ digested with EcoRI and XbaI. 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 G_i. 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 ₂AR-G_i2 fusion protein (used as immunoblotting standard for the determination of G_i2 expression) was prepared by overlap extension PCR analogous to the FPR-G_i 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 (25, 41). Sf9 cells were co-infected with recombinant baculoviruses encoding nonfused FPR, G_i2, and G protein ₁₂ complex or fusion proteins plus G protein ₁₂ complex.

Analysis of FPR-G_i Fusion Proteins by Agonist Saturation Binding, GTPS Binding, and GTPase Activity-- [³H]fMLF saturation binding, GTPS saturation binding, and time course of GTPS binding in Sf9 membranes expressing FPR-G_i fusion proteins plus ₁₂ complex were performed exactly as described for Sf9 membranes expressing nonfused FPR, G_i2, and ₁₂ complex (25). Steady-state GTPase activity with different substrate concentrations was determined as described for Sf9 membranes expressing ₂AR-G_sL fusion protein except that the MgCl₂ concentration was 5 mM instead of 1 mM (41).

Photoaffinity Labeling of FPR-G_i Fusion Proteins-- AA-GTP was prepared from [-³²P]GTP as described (47). Labeling of Sf9 membranes was performed essentially as described (47). Briefly, Sf9 membranes expressing FPR-G_i 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 mM MgCl₂, 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-G_i 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 ₂AR (25, 41), anti-His₆ Ig, which recognizes the C-terminal His₆ epitope of the FPR and ₂AR (25, 41), anti-G_i1/2 Ig, and anti-G_i3 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 (25).

Analysis of FPR-G_i 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 G_i1 and G_i2 in FPR-G_i 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.

Miscellaneous-- Protein was determined using the Bio-Rad DC protein assay kit. Data were analyzed by nonlinear regression, using the Prism program.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of FPR-G_i Fusion Protein Expression on the Protein and mRNA Level-- The human FPR expressed in Sf9 cells has a molecular mass of 40 kDa (25), and the molecular mass of G_i proteins is 40-41 kDa (11, 13). Thus, the expected molecular mass of FPR-G_i fusion proteins is 80-81 kDa. Indeed, the M1 antibody, which recognizes the FLAG epitope attached to the N terminus of the FPR (25) detected antigens of the appropriate mass in membranes prepared from baculovirus-infected Sf9 cells (Fig. 1A). The anti-G_i1/2 Ig detected the FPR-G_i1 and FPR-G_i2 fusion proteins but not FPR-G_i3 (Fig. 1B). In contrast, the anti-G_i3 Ig reacted with the FPR-G_i3 fusion protein but not with FPR-G_i1 or FPR-G_i2 (Fig. 1C). There was no indication for proteolytic degradation of fusion proteins. Because the anti-G_i1/2 Ig cannot discriminate between G_i1 and G_i2 (Fig. 1B), we performed reverse transcriptase-PCR analysis on mRNA from Sf9 cells infected with the FPR-G_i1 and FPR-G_i2 baculoviruses to differentiate between the two G_i isoforms. G_i1 has a unique SacI site at position 382, and G_i2 has a unique BamHI site at position 653 (Fig. 1D). The G_i portions of fusion proteins were amplified by PCR and digested. SacI generated the expected 382- and 711-bp fragments with G_i1, whereas G_i2 cDNA was not cut. In contrast, digestion with BamHI yielded the expected 443- and 653-bp fragments with G_i2, whereas G_i1 cDNA was not cut. Taken together, the immunoblotting and reverse transcriptase-PCR data document the specific expression of FPR-G_i1, FPR-G_i2, and FPR-G_i3 fusion proteins in Sf9 cell membranes.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Analysis of the expression of FPR-Gi fusion proteins in Sf9 cells. A-C, membranes from Sf9 cells expressing FPR-Gi fusion proteins at ~1.2-1.4 pmol/mg as assessed by [3H]fMLF saturation binding were prepared, separated by SDS-PAGE, and probed with anti-FLAG Ig (A), anti-Gi1/2 Ig (B), and anti-Gi3 Ig (C) as described under "Experimental Procedures." Numbers on the left indicate molecular masses of marker proteins. Shown are the horseradish peroxidase-reacted nitrocellulose membranes of gels containing 10% (w/v) acrylamide. D, mRNA from Sf9 cells infected with FPR-Gi1 and FPR-Gi2 baculovirus, respectively, was isolated and reverse-transcribed as described under "Experimental Procedures." The Gi portions of fusion proteins were amplified by PCR and digested with SacI or BamHI. Digested DNA was separated on gels containing 2% (w/v) agarose. The left lanes of the gels show the 100-bp DNA ladder. The broad calibration lanes represent the 500- and 1000-bp standards. The scheme on the left shows the relative positions of the SacI site in Gi1 and the BamHI site in Gi2.

[³H]fMLF Binding Studies-- Fig. 2 (A and B) shows representative [³H]fMLF saturation binding curves for FPR-G_i1, and Table I summarizes the data for all three fusion proteins. For comparison, the [³H]fMLF binding data for nonfused FPR co-expressed with G_i2 are included as well in Table I. FPR-G_i fusion proteins bound the agonist [³H]fMLF (0.2-30 nM) according to single-site saturation curves. The K_d and B_max values of [³H]fMLF binding to FPR-G_i1, FPR-G_i2, and FPR-G_i3 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 increase B_max 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 GTPS to G uncouples GPCRs from G proteins and reduces high affinity agonist binding (7, 25, 48). By analogy to nonfused FPR co-expressed with G_i2, GTPS substantially reduced the B_max of [³H]fMLF binding to FPR-G_i fusion proteins and, to a variable extent, increased the K_d for [³H]fMLF.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Functional analysis of the FPR-Gi1 fusion protein. Sf9 membranes expressing FPR-Gi2 at 1.0-2.2 pmol/mg as assessed by [3H]fMLF saturation binding were subjected to various analyses as described under "Experimental Procedures." A and B, [3H]fMLF saturation binding. Reaction mixtures contained Sf9 membranes, [3H]fMLF at the concentrations indicated on the abscissa and solvent (control) or GTPS (10 µM). A shows the specific binding in the absence and presence of GTPS, i.e. the binding not competed for by 10 µM unlabeled fMLF. B shows the difference between [3H]fMLF binding in the absence and presence of 10 µM GTPS. C, time course of GTPS binding. Reaction mixtures contained Sf9 membranes, 1 nM [35S]GTPS plus 9 nM unlabeled GTPS, 1 µM GDP, and solvent (basal), fMLF (10 µM), or CsH (10 µM). Reactions were stopped at the time points indicated on the abscissa. D, effects of fMLF and CsH on GTPS saturation binding. Reaction mixtures contained Sf9 membranes, 0.1-1 nM [35S]GTPS plus unlabeled GTPS 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). For each GTPS concentration, the basal GTPS binding was subtracted from the GTPS binding value observed in the presence of fMLF to calculate the increase in GTPS binding caused by fMLF. From each basal GTPS concentration, the GTPS binding value observed in the presence of CsH was subtracted to obtain the decrease of GTPS binding caused by CsH. The dotted line is the extrapolation of basal GTPS binding. E, concentration-response curves for various FPR ligands on steady-state GTPase activity. Reaction mixtures contained Sf9 membranes, 100 nM [-32P]GTP, and solvent (2% v/v Me2SO) or ligands at various concentrations. F, kinetics of steady-state GTP hydrolysis. Reaction mixtures contained Sf9 membranes, 30 nM-1.5 µM [-32P]GTP 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 of 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. GTPase activities were divided by Bmax values of ligand-regulated GTPS binding (Bmax total) to obtain molar turnover numbers. The dotted line is the extrapolation of basal GTPase activity. The Fig. shows the means ± S.D. of representative experiments performed intriplicate. Summaries of the data for FPR-Gi1 and the other fusion proteins as well as the co-expression system consisting of nonfused FPR plus Gi2 are given in Tables I-IV. Tables I-IV also provide details on the specific type of nonlinear regression analysis performed with each data set.

View this table: [in this window] [in a new window]   Table I [3H]fMLF saturation binding in Sf9 membranes expressing FPR-Gi fusion proteins: comparison with nonfused FPR co-expressed with Gi2 Membranes from Sf9 membranes expressing FPR-Gi 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 GTPS. Nonspecific binding, i.e. the [3H]fMLF binding not competed for by 10 µM unlabeled 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 Gi2 were taken from Ref. 25.

Kinetics of GTPS Binding-- Ligand-regulated GTPS 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 (22, 25, 49). Fig. 2C shows a representative time course experiment for the GTPS binding to FPR-G_i1, and Fig. 2D shows a representative GTPS saturation binding experiment for FPR-G_i1. Table II summarizes the GTPS binding data for all three fusion proteins. For comparison, the GTPS binding data for the FPR co-expressed with G_i2 are included in Table II as well. As reported previously for the FPR co-expressed with G_i2 in Sf9 cells (25), the FPR fused to G_i2 displayed high constitutive activity as assessed by the strong inhibitory effect of the inverse agonist CsH on GTPS binding. CsH also displayed strong inhibitory effects at the FPR fused to G_i1 and G_i3. The K_d values for fMLF-stimulated and CsH-inhibited GTPS binding were similar for all FPR-G_i fusion proteins studied and compare favorably with the K_d values for GTPS binding with nonfused FPR co-expressed with G_i2. With respect to the time course of GTPS binding, fMLF reduced whereas CsH increased t_1/2, but there was some variability between the different systems studied.

View this table: [in this window] [in a new window]   Table II Effects of fMLF and CsH on [35S]GTPS binding in Sf9 membranes expressing FPR-Gi fusion proteins and nonfused FPR with Gi2: saturation binding and time course Membranes from Sf9 cells expressing FPR-Gi fusion proteins were prepared. For saturation binding experiments, membranes were incubated for 60 min in the presence of 0.1-1 nM [35S]GTPS plus unlabeled GTPS 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 GTPS. For each GTPS concentration, the basal GTPS binding value was subtracted from the GTPS binding value observed in the presence of fMLF to calculate the increase in GTPS binding caused by fMLF. From each basal GTPS binding value, the GTPS binding value observed in the presence of CsH was subtracted to obtain the decrease of GTPS 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]GTPS plus 9 nM unlabeled GTPS, 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 Gi2 were taken from Ref. 25.

Because there is a defined 1:1 stoichiometry of GPCR to G in fusion proteins (7, 41-43), 1 mol of activated GPCR can maximally stimulate the binding of 1 mol of GTPS to its fused G partner. Thus, one would expect that the B_max of receptor ligand binding is very similar to the B_max of ligand-regulated GTPS binding. The B_max of ligand-regulated GTPS binding (B_max total) is the difference between maximum GTPS binding stimulated by fMLF and minimum GTPS binding inhibited by CsH. As is evident from the comparison of B_max values for [³H]fMLF binding and B_max total values of GTPS binding (compare Tables I and II and Fig. 2, A, B, and D), B_max total of GTPS binding surpasses B_max of [³H]fMLF binding by 4.7-fold for FPR-G_i1, by 5.9-fold for FPR-G_i2, and by 2.5-fold for FPR-G_i3.

Resolution of the Discrepancies between the B_max of [³H]fMLF Binding and Ligand-regulated GTPS Binding-- Two explanations for the discrepancies between the B_max values of [³H]fMLF binding and ligand-regulated GTPS binding have to be considered. First, the fused FPR could interact not only with its fused G_i partner but also with G proteins of the host cell. Indeed, cross-talk of a fused G_i protein-coupled GPCR to endogenous G proteins occurs in some mammalian expression systems (50, 51). Second, the FPR could still couple only to its fused G 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 GTPS binding assay because the receptor promotes binding of GTPS 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 (48, 52).

To address the first explanation, we visualized FPR-activated G proteins by photoaffinity labeling with AA-GTP (12, 13, 47). 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 (25). fMLF efficiently stimulated the incorporation of AA-GTP into all three FPR-G_i 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 (46). 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 (25, 53).

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Photoaffinity labeling of proteins in membranes expressing FPR-Gi fusion proteins by AA-GTP. Membranes from Sf9 cells expressing FPR-Gi fusion proteins at ~1.2-1.4 pmol/mg as assessed by [3H]fMLF saturation binding were prepared and incubated with 1 µCi of AA-GTP in the presence of solvent (control, ) or 10 µM fMLF (+) under the conditions described under "Experimental Procedures." Proteins were irradiated with UV light and separated by SDS-PAGE. Numbers on the left indicate molecular masses of marker proteins. Shown is the autoradiograph of a gel containing 8% (w/v) acrylamide. Similar results were obtained in three independent experiments.

To address the second explanation, we determined the expression of FPR-G_i 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") (41, 43). The previously characterized ₂AR-G_sL fusion protein can be used as standard to assess the expression level of other proteins bearing the same epitopes because the expression level of ₂AR-G_sL can be unequivocally determined by receptor antagonist saturation binding (41, 43). Fig. 4A shows that the immunoreactivity of FPR-G_i1 expressed at 1.4 pmol/mg ([³H]fMLF saturation binding) was even higher than the immunoreactivity of ₂AR-G_sL expressed at 8.6 pmol/mg ([³H]DHA saturation binding) using the anti-His₆ Ig. The signals with FPR-G_i2 (Fig. 4A) and FPR-G_i3 (data not shown) expressed at 1.0 pmol/mg each were slightly smaller than the signals obtained with ₂AR-G_sL expressed at 8.6 pmol/mg. By using the anti-FLAG Ig, the immunoreactivity with ₂AR-G_sL expressed at 8.6 pmol/mg was moderately higher than with FPR-G_i1 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 (25, 53, 54) and that this glycosylation could interfere with the recognition of the N-terminal FLAG epitope by the M1 antibody.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Analysis of the expression level of FPR-Gi fusion proteins and nonfused FPR by quantitative immunoblotting. Sf9 membranes expressing 2AR-GsL, FPR-Gi fusion proteins, nonfused 2AR, and nonfused FPR were prepared. The expression level of 2AR-GsL and 2AR was determined by antagonist saturation binding ([3H]DHA being the radioligand). The expression level of FPR-Gi fusion proteins and nonfused FPR was determined by agonist saturation binding ([3H]fMLF being the radioligand). Expression levels of membranes are shown. Defined amounts of protein (expressed as µg/lane) from membranes expressing the specific fusion proteins and receptors were separated by SDS-PAGE and probed with various antibodies as described under "Experimental Procedures." A, analysis of 2AR-GsL and FPR-Gi fusion proteins with anti-His6 Ig. B, analysis of 2AR-GsL and FPR-Gi1 with anti-FLAG Ig. C, analysis of nonfused 2AR and FPR with anti-FLAG Ig. Numbers on the left indicate molecular masses of marker proteins. Shown are the horseradish peroxidase-reacted nitrocellulose membranes of gels containing 10% (w/v) acrylamide. Similar results were obtained in three independent experiments.

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 ₂AR or the FPR plus G_i2 plus ₁₂ complex using the anti-FLAG Ig. Fig. 4C shows that the FPR runs as a much more diffuse band in SDS-PAGE than the ₂AR, indicating that the FPR is more heavily glycosylated than the ₂AR. Despite this difference in glycosylation pattern of the two GPCRs, it is evident that the immunoreactivity of the ₂AR 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 GTPS saturation binding studies (Tables I and II). The similarity of the fusion protein expression levels as determined by immunoblotting and GTPS saturation binding also implies that the majority, if not all, of the expressed fusion protein molecules are functionally active. Of interest, by comparing the B_max values of agonist and antagonist binding at the adenosine A₁ receptor co-expressed with, or fused to, G_i proteins, it was also shown that agonist binding underestimates GPCR expression level by ~3-6-fold (55). Unfortunately, an antagonist radioligand for the FPR is not available, but the combination of immunoblotting and ligand-regulated GTPS binding can compensate for this deficiency.

We also assessed the potency of fMLF at activating the fused G_i partner. If the FPR in a state of low agonist affinity does indeed couple to G_i proteins, then the EC₅₀ values for fMLF in functional assays should be considerably higher than the K_d 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 K_d values for high affinity [³H]fMLF binding (compare Fig. 2 (A, B, and E) and Tables I and III). A similar discrepancy between agonist affinity and agonist potency was reported for nonfused FPR expressed in HL-60 leukemia cells and Sf9 cells (25, 26).

View this table: [in this window] [in a new window]   Table III Steady-state GTPase activity in Sf9 membranes expressing FPR-Gi fusion proteins: efficacy and potency of FPR ligands Membranes expressing FPR-Gi fusion proteins were incubated in the presence of 100 nM [-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).

GTP Turnover Measurements-- The defined 1:1 stoichiometry of GPCR and G in fusion proteins allows determination of ligand-regulated GTP turnover in a membrane system (41, 42). Specifically, GTPase activities are divided by B_max values of receptor antagonist saturation binding to calculate ligand-regulated GTP turnover in fusion proteins (41, 42). However, because agonist saturation binding largely underestimates the actual expression level of FPR-G_i fusion proteins (Tables I and II and Figs. 2, A, B, and D, and 4), we divided GTPase activities by the B_max values of ligand-regulated GTPS binding. Fig. 2F shows a representative experiment for the kinetics of ligand-regulated GTP turnover at FPR-G_i1, and Table IV summarizes the data for all three fusion proteins. The K_m values of fMLF-stimulated GTP hydrolysis for FPR-G_i fusion proteins were similar to each other as were the K_m values for CsH-inhibited GTP hydrolysis. The V_max values of ligand-regulated GTP turnover were similar for the three FPR-G_i fusion proteins. The kinetic parameters of the GTPase of FPR-G_i fusion proteins are also similar to the corresponding parameters of an ₂-adrenoreceptor-G_i1 fusion protein (42).

View this table: [in this window] [in a new window]   Table IV Kinetics of steady-state GTP hydrolysis in Sf9 membranes expressing FPR-Gi fusion proteins Membranes expressing FPR-Gi fusion proteins were incubated in the presence of 30 nM to 1.5 µM [-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 GTPS binding of the respective membrane (see Table II) 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.

Determination of Agonist Efficacies and Potencies-- The data obtained with the full agonist fMLF and the full inverse agonist CsH in GTPS binding and GTPase studies (Tables II-IV) point to high constitutive activity of the FPR coupled to all G_i isoforms. We wished to explore the hypothesis that there are, nonetheless, subtle differences in the constitutive activity of the FPR coupled to G_i 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 (7, 56, 57). The GTPase assay is particularly suitable for determination of ligand efficacies and potencies because it monitors, unlike the GTPS binding assay, receptor-G protein coupling at steady state (7, 41). Moreover, the GTPase assay has already successfully been used to dissect differences in the constitutive activity of a given receptor coupled to different G isoforms (7).

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-G_i1, and Table III provides a summary for all three FPR-G_i 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-G_i2 than at FPR-G_i1 and FPR-G_i3.

Expression Level of Nonfused G_i2 in the Co-expression System-- When the FPR-G_i2 fusion protein was compared with the corresponding co-expression system, we were puzzled that not only the B_max values of [³H]fMLF binding were similar to each other but also the B_max values of ligand-regulated GTPS binding (compare Tables I and II). However, if the FPR had activated G_i catalytically in the co-expression system, we would have expected a much higher B_max of ligand-regulated GTPS binding in this system than in the fusion protein system. Thus, we had to address the hypothesis that the low B_max of ligand-regulated GTPS binding in the co-expression system was due to a low expression level of G_i proteins, resulting in poor availability of G proteins for activation by GPCR. We used a ₂AR-G_i2 fusion protein expressed at 15 pmol/mg ([³H]DHA saturation binding) as standard for analyzing the expression level of nonfused G_i2. The immunoreactivity with the anti-G_i1/2 Ig in membranes expressing FPR plus G_i2 was ~20-30 times higher than the immunoreactivity with similar amounts of protein from membranes expressing ₂AR-G_i2. Thus, in the co-expression system, G_i2 is expressed at a level of ~300-450 pmol/mg. Similar expression levels in Sf9 cells were also reported for other G proteins (41, 58). 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 G_i2 cannot account for the surprisingly low B_max of ligand-regulated GTPS binding in this system.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Highly Efficient Receptor-G Protein Coupling in a FPR-G_i2 Fusion Protein-- The aim of our study was to quantitatively analyze the coupling of the FPR to the three G_i 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 G isoforms (7, 43). However, because the fusion of a receptor C terminus to the G 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, G_i protein-coupled receptors cannot only couple to their fused G_i partner but also to the endogenous G proteins of the host cells (50, 51). To address these concerns, we compared the coupling of the FPR to G_i2 in the fused and nonfused state and visualized activated G proteins by photoaffinity labeling. There were only minor differences in FPR-G_i2 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 G_i isoforms.

In the co-expression system, there is an ~100-fold molar excess of G_i2 over FPR as assessed by quantitative immunoblotting with defined standards (Figs. 4C and 5). Despite the large excess of G protein relative to receptor in the co-expression system and the high absolute expression levels of FPR and G_i2, the signaling efficiency of the co-expression system as assessed by the B_max of ligand-regulated GTPS 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 G_i relative to the FPR. Similar conclusions were obtained for ₂AR-G_s and adenosine A₁ receptor-G_i fusion proteins (41, 55, 59).

View larger version (72K): [in this window] [in a new window]   Fig. 5.   Analysis of the expression level of Gi2 in the co-expression system consisting of FPR and Gi2 by quantitative immunoblotting. Sf9 membranes expressing 2AR-Gi2 and nonfused FPR plus Gi2 were prepared. The expression level of 2AR-Gi2 was determined by antagonist saturation binding ([3H]DHA being the radioligand) and is indicated in the figure. Defined amounts of protein (expressed as µg/lane) from membranes expressing 2AR-Gi2 or nonfused FPR plus Gi2 were separated by SDS-PAGE and probed with anti-Gi1/2 Ig as described under "Experimental Procedures." Numbers on the left indicate molecular masses of marker proteins. Shown are the horseradish peroxidase-reacted nitrocellulose membranes of gels containing 10% (w/v) acrylamide. Similar results were obtained in three independent experiments.

Similarly Efficient Coupling of the FPR to G_i1, G_i2, and G_i3-- 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 G_i2 is lower than the GDP affinity of G_i1 and G_i3 (37, 38). In addition, the FPR co-expressed with G_i2 has the properties of a constitutively active GPCR (25). Moreover, G_sL has a lower GDP affinity than G_sS, and the ₂AR coupled to G_sL is constitutively active, too (7, 39). An explanation for these findings could be that the lower the GDP affinity of a given G 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 G_i2 would have a higher degree of constitutive activity than the FPR coupled to G_i1 and G_i3. Surprisingly, however, the relative inhibitory effects of CsH at the total ligand-regulated GTPS binding and GTP hydrolysis were very similar among the three G_i isoforms (Tables II-IV). Another indicator for increased constitutive activity of a GPCR is elevated efficacy and potency of partial agonists (7, 56, 57). However, we could not detect consistent increases in these parameters for FPR-G_i2 as compared with FPR-G_i1 and FPR-G_i3 (Table IV). Taken together, our data show that the different G_i 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 G_s splice variants to the ₂AR 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 G_i isoforms is too subtle to become effective.

The lack of substantial differences in the coupling of the FPR to G_i isoforms is somewhat unexpected in view of the fact that many GPCRs show differences in coupling efficiency to G_i isoforms, that G_i2 is the major G protein in phagocytes, and that G_i1 is not expressed in these cells (27, 29, 31-34). Of interest, the first, second, and third intracellular loops as well as the C terminus of the FPR are involved in G protein coupling (9, 60), whereas for most other receptors, the areas involved in G protein coupling appear to be more confined (61-63). Thus, the extensive contact area of the FPR with G proteins may provide the structural basis for the lack of differential coupling to G_i isoforms. Based on our findings, one can assume that in vivo, G_i2 and G_i3 are used interchangeably by the FPR.

Stoichiometry of FPR-G_i Coupling: Implications for Signaling in Vivo-- Because FPR-G_i fusion proteins have a defined 1:1 stoichiometry of GPCR and G and because there is no cross-talk of the fused receptor with endogenous G proteins, we expected very similar B_max values for [³H]fMLF binding and ligand-regulated GTPS binding, reflecting the fact that one FPR molecule can activate one G_i molecule. However, the B_{max total} values of GTPS binding were up to 6-fold higher than the B_max 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 EC₅₀ values of fMLF at activating GTP hydrolysis and guanine nucleotide binding to G_i in fused and nonfused systems are ~100-fold higher than the K_d values for [³H]fMLF binding (12, 13, 25, 26). Additionally, quantitative immunoblotting with anti-His₆ Ig and anti-FLAG Ig using ₂AR-G_sL as standard demonstrated that agonist saturation binding greatly underestimates the actual expression level of FPR fusion proteins (Fig. 4, A and 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-G_i protein coupling in nonfused systems. We and other groups had proposed that the FPR activates G_i proteins catalytically, i.e. a single FPR molecule activates several G_i