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J Biol Chem, Vol. 273, Issue 37, 24181-24189, September 11, 1998


High Constitutive Activity of the Human Formyl Peptide Receptor*

Katharina Wenzel-SeifertDagger §, Carl M. Hurtparallel , and Roland SeifertDagger §

From the Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305-5428

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent ("basal") Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist, i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Gialpha 2beta 1gamma 2 reconstituted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) binding and GTPgamma S-sensitive high affinity [3H]FMLP binding. The FPR "antagonist" cyclosporin H (CsH) potently and efficiently reduced basal GTPgamma S binding in Sf9 membranes. Another FPR antagonist, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L-phenylalanine did not inhibit basal GTPgamma S binding but blocked the inhibitory effect of CsH on GTPgamma S binding. Na+ reduced basal GTPgamma S binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Human neutrophils play a key role in host defense against bacterial infections (1-3). Neutrophils are activated by the bacterial formyl peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP).1 FMLP binds to a specific formyl peptide receptor (FPR) that initiates a complex signaling cascacde ultimately resulting in chemotaxis and the release of cytotoxic products such as reactive oxygen species and lysosomal enzymes (1-3). The FPR belongs to the chemoattractant receptor family that is part of the superfamily of G-protein-coupled receptors (GPCRs) (4, 5). The FPR couples to the pertussis toxin (PTX)-sensitive G-proteins Gi2 and Gi3 (6, 7). The stimulatory effects of FMLP are blocked by the competitive FPR antagonists N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L-phenylalanine (BocPLPLP) and cyclosporin H (CsH) (8-11). BocPLPLP is a synthetic linear peptide analogue of FMLP (9), and CsH is a cyclic undecapeptide from the fungus Tolyplocadium inflatum Gams (12). Chemically, CsH is a stereoisomer of the immunosuppressant drug cyclosporin A (13), but cyclosporin A is not an FPR antagonist (10, 11).

The two-state model of GPCR activation assumes that GPCRs exist in an inactive ("R") state or an active ("R*") state (14-16). According to this model, the isomerization of GPCR from R to R* can occur, to some extent, also in the absence of agonist which process is referred to as constitutive activity. Agonists stabilize the R* state and increase basal G-protein activity. However, since GPCRs can isomerize from R to R* even agonist-independently, the basal G-protein activity actually does not reflect the intrinsic basal activity of the G-protein but rather basal GPCR activity. Inverse agonists stabilize the R state and decrease basal G-protein activity. Neutral antagonists do not affect the equilibrium between R and R* and block both agonist and inverse agonist effects. In addition to agonists, G-proteins can stabilize the R* state (17, 18).

Many wild-type GPCRs including the beta 2-adrenoreceptor (19-21), the 5-hydroxytryptamine2C receptor (22), the histamine H2 receptor (23), the delta -opioid receptor (24), the thyrotropin receptor (25) and the alpha 2D-adrenoreceptor (26) are constitutively active. The absolute expression levels of GPCR and G-proteins and their relative stoichiometry to each other determine the sensitivity with which constitutive activity is detected. Specifically, at a low GPCR expression level, a given compound may appear to act as neutral antagonist, whereas at high GPCR and/or G-protein expression levels, the compound may behave as inverse agonist (17, 20, 21, 27, 28).

Circumstantial evidence suggests that the FPR is constitutively active as well. In particular, PTX inhibits not only the FMLP-induced Gi-protein activation but also reduces the basal Gi-protein activity in membranes of myeloid differentiated HL-60 cells (29-31). Additionally, Na+ efficiently reduces basal G-protein activity in membranes expressing Gi-protein-linked constitutively active GPCRs (26, 32) and in myeloid membranes expressing FPR (29, 30).

We hoped to identify an inverse FPR agonist to validate the assumed constitutive activity of the FPR. To achieve this aim, we took advantage of the Spodoptera frugiperda (Sf9) insect cell expression system. The Sf9 cell system provides a low background of endogenous G-proteins (17, 33, 34) and is highly sensitive at unmasking constitutive activity of GPCRs, provided that the appropriate complement of G-proteins is expressed (17, 20, 21, 35-37). Ye and colleagues (37) have already shown that the human FPR can be expressed in Sf9 cells. Of interest, Sf9 cells do not endogenously express Gi-proteins to efficiently support G-protein-coupling of the FPR. Therefore, we co-expressed the human FPR with the mammalian G-protein, Gialpha 2beta 1gamma 2, in Sf9 cells. We also expressed the FPR in human embryonic kidney (HEK293) cells. These cells endogenously express Gi-proteins to which exogenously expressed chemoattractant receptors can couple (38, 39). As read-out for FPR activity, we monitored guanosine 5'-O-(3-thiotriposphate) (GTPgamma S) binding to G-proteins (30, 35, 36).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The cDNA of FPR-26 in pCDM8 was kindly provided by Dr. F. Boulay (Laboratoire de Biochimie, CNRS, Grenoble, France) (40). Recombinant baculovirus encoding the unmodified versions of the G-protein subunits beta 1gamma 2 was a kind gift of Dr. P. Gierschik (Abteilung für Pharmakologie und Toxikologie, Universität Ulm, Germany) (41). Recombinant baculovirus encoding for Gialpha 2 was donated by Dr. A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Antibodies recognizing G-protein beta -subunits (anti-Gbeta common, AS 11) (42) and Gialpha -subunits (anti-Gialpha common, AS 266) (34) were generously provided by Drs. B. Nürnberg and G. Schultz (Institut für Pharmakologie, Freie Universität Berlin, Germany). CsH was kindly provided by Novartis (Basel, Switzerland). BocPLPLP and FMLP were from Sigma. Stock solutions of CsH and BocPLPLP (1 mM each) and FMLP (10 mM) were prepared in Me2SO and were stored at -20 °C under light protection. Dilutions of FPR ligands were made fresh daily in distilled water. The final Me2SO concentration in a given experiment was held constant (0.2-2.0% (v/v) Me2SO, depending on the particular experiment performed). [35S]GTPgamma S (1000-1500 Ci/mmol) and [3H]FMLP (56 Ci/mmol) were from NEN Life Science Products. The M1 antibody was from IBI (New Haven, CT). Unlabeled GTPgamma S and GDP were obtained from Boehringer Mannheim (Mannheim, Germany). Glass fiber filters (GF/C) were from Schleicher & Schuell (Dassel, Germany). PTX was from List Biological Laboratories (Campbell, CA). All other reagents were of the highest purity available and were from standard suppliers.

Construction of FLAG Epitope- and Hexahistidine-tagged Modified FPR-DNA-- The FPR, like most GPCRs, is a type IIIb membrane protein with an extracellular N terminus without a signal sequence. Adding a signal sequence to the N terminus serves to direct the membrane insertion of proteins cotranslationally (43) and enhances the production of functional GPCR protein in insect cells (44). Therefore, a DNA sequence encoding the cleavable signal peptide from influenza hemagglutinin followed by the FLAG epitope, which can be recognized by the M1 antibody, was placed 5' of the start codon of the DNA of FPR-26. We also added a hexahistidine tag to the FPR C terminus to allow future purification of the receptor and to provide additional protection against proteolysis (45, 46). The FPR modifications were generated by sequential overlap-extension PCRs using Pfu polymerase (Stratagene, La Jolla, CA). In PCR 1A, the DNA sequence of the signal peptide (S) and the FLAG epitope (F) was amplified with pGEM-3Z-SF-hbeta 2-adrenoreceptor-6His (20) as template by using a sense primer 5' of the SacI site near the SF sequence (sense pGEM primer) and an antisense primer encoding the last 19 bp of the SF. In PCR 1B, the cDNA of FPR-26 was amplified using pCDM8-FPR-26 as template with a sense primer which annealed with the first 17 bp of the 5'-end of the FPR and included the last 19 bp of the SF in its 5'-extension. The antisense primer encoded amino acids 331-348 of the FPR with a silent mutation in the triplet of Thr-336 (ACC right-arrow ACG). Thereby, we introduced a unique EcoRI site for diagnostic purposes and future cloning procedures. In PCR 2, the products of PCRs 1A and B were used as templates. The sense pGEM primer and an antisense primer, annealing with the last 15 bp of the 3'-end of PCR product 1B and having an extension that encodes the two C-terminal amino acids of FPR-26, a hexahistidine tag, the stop codon, and an extra XbaI site for cloning purposes, were used to prime PCR 2. In this way, a fragment encoding the signal sequence, the FLAG epitope, FPR-26 cDNA, and a hexahistidine tag followed by an XbaI site was obtained. This fragment was digested with SacI and XbaI and cloned into pGEM-3Z-SF-hbeta 2-adrenoreceptor-6His digested with SacI and SalI together with a linker oligonucleotide encoding (5' to 3') an XbaI site, a BamHI site, and a SalI site. PCR-generated DNA sequences were confirmed by enzymatic sequencing using Sequenase version 2.0 Sequencing kit (U. S. Biochemical Corp.). The cDNA encoding the FLAG epitope- and hexahistidine-tagged FPR was cloned into the baculovirus expression vector pVL 1392 and the mammalian expression vector pcDNA 3.0 using the HindIII site at the 5'-end of the SF region and the XbaI site at the 3'-end of the receptor.

Generation of Recombinant Baculoviruses and Cell Culture and Membrane Preparation-- Recombinant baculovirus encoding FPR was generated in Sf9 cells using the BaculoGold transfection kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. After initial transfection, working virus stocks were generated by three sequential virus amplifications. Sf9 cells were cultured in 250-ml disposable Erlenmeyer flasks at 28 °C under rotation at 125 rpm in SF 900 II medium (Life Technologies, Inc.) supplemented with 5% (v/v) fetal calf serum (Gemini, Calabasa, CA) and 0.1 mg/ml gentamicin (Boehringer Mannheim, Mannheim, Germany). Cells were maintained at a density of 0.5-6.0 × 106 cells/ml. For infection, cells were sedimented by centrifugation and suspended in fresh medium. Cells were seeded at 3.0 × 106 cells and infected with a 1:100 dilution of baculovirus stocks. Cells were cultured for 48 h before membrane preparation.

HEK293 cells were cultured in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum at 37 °C and 5% (v/v) CO2. Cells were transfected with pcDNA 3.0 encoding human FPR by calcium phosphate precipitation. For selection of stably transfected cells, 0.5 mg/ml genticin (Life Technologies, Inc.) was added to the culture medium following transfection (47). Antibiotic-resistant clones were isolated and assayed for expression of FPR by [3H]FMLP binding (see below).

Sf9- and HEK293 membranes were prepared as described (33) using 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine, and 10 µg/ml leupeptin as protease inhibitors.

[3H]FMLP Binding-- Before experiments, membranes were pelleted by a 15-min centrifugation at 4 °C and 15,000 × g and resuspended in binding buffer (1 mM EDTA, 12.5 mM MgCl2, and 75 mM Tris/HCl, pH 7.4). For determination of Kd and Bmax values, reaction mixtures (500 µl) contained membranes (20-60 µg of protein/tube) in binding buffer supplemented with [3H]FMLP (0.2-30 nM). Nonspecific binding was determined in the presence of [3H]FMLP (0.2-30 nM) plus 10 µM unlabeled FMLP. Incubations were performed for 60 min at 25 °C and shaking at 200 rpm. Reaction mixtures additionally contained solvent (basal) or GTPgamma S (10 µM). In competition studies, tubes contained 100 µg of membrane protein, 5 nM [3H]FMLP, and non-labeled BocPLPLP or CsH at various concentrations. Nonspecific [3H]FMLP binding was less than 5% of total binding. Bound [3H]FMLP was separated from free [3H]FMLP by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4 °C). Filter-bound radioactivity was determined by liquid scintillation counting.

[35S]GTPgamma S Binding-- Before experiments, membranes were pelleted by a 15-min centrifugation at 4 °C and 15,000 × g. Membranes were resuspended in binding buffer. Reaction mixtures (500 µl) contained membranes (12-25 µg of protein/tube) in binding buffer supplemented with 0.05% (w/v) BSA, 0.4 nM [35S]GTPgamma S, and 1 µM GDP in the presence of different concentrations of FPR ligands. In some experiments, reaction mixtures additionally contained NaCl and KCl at various concentrations. GTPgamma S saturation binding studies were performed in the presence of 1 µM GDP and 1 nM [35S]GTPgamma S, and various concentrations of unlabeled GTPgamma S to give various final ligand concentrations. Incubations were performed for 45 min at 25 °C and shaking at 200 rpm. For time course studies, Sf9 membranes (16 µg of protein/tube) were suspended in 1500 µl of binding buffer supplemented with 1 nM [35S]GTPgamma S plus 4 nM unlabeled GTPgamma S, 1 µM GDP, and FPR ligands at a fixed concentration or solvent (basal). Aliquots of 200 µl were taken at seven different time points. Nonspecific [35S]GTPgamma S binding was determined in the presence of 10 µM GTPgamma S and was less than 0.1% of total binding. Bound [35S]GTPgamma S was separated from free [35S]GTPgamma S by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4 °C). Filter-bound radioactivity was determined by liquid scintillation counting.

SDS-PAGE and Immunoblot Analysis-- SDS-PAGE was performed according to Laemmli (48). Solubilized membrane proteins were separated on a gel containing 10% (w/v) acrylamide. Immunoblotting was performed according to Towbin et al. (49). Nitrocellulose membranes were reacted with M1 antibody (1: 1000), anti-Gialpha common antibody (1: 200), or anti-Gbeta common antibody (1: 200). Immunoreactive bands were visualized by sheep anti-mouse IgG (M1 antibody) and donkey anti-rabbit IgG (anti-Gialpha common and anti-Gbeta common antibody), respectively, coupled to peroxidase, using o-dianisidine and H2O2 as substrates.

Miscellaneous-- Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad). Data were analyzed by non-linear regression, using the Prism program (GraphPad, Prism, San Diego, CA).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Co-expression of FPR with Gialpha 2beta 1gamma 2 in Sf9 Membranes-- Membranes from uninfected Sf9 cells and Sf9 cells triple-infected with FPR-, Gialpha 2-, and beta 1gamma 2-encoding baculoviruses were prepared and subjected to immunological analysis with the M1 antibody, recognizing the N-terminal FLAG epitope of the FPR, anti-Gialpha common antibody, and anti-Gbeta common antibody. In baculovirus-infected Sf9 membranes, the M1 antibody recognized a broad and diffuse band with an apparent molecular mass of ~40 kDa, consistent with the expression of glycosylated FPR (Fig. 1A). In uninfected Sf9 cells, no immunoreactivity with the M1 antibody was detected. The migration of the non-modified FPR expressed in Sf9 cells in SDS-PAGE (37) is similar to the migration of the FLAG epitope- and hexahistidine-tagged FPR. The FPR expressed in neutrophil membranes migrates as 50-70-kDa protein in SDS-PAGE, indicating that glycosylation of the FPR in Sf9 cells is less extensive than in mammalian cells (50).


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  		Fig. 1.   Immunological characterization of FPR, Gialpha 2, and Gbeta 1-subunit in Sf9 membranes. Membranes from uninfected Sf9 cells and Sf9 cells expressing FPR plus Gialpha 2beta 1gamma 2 (50 µg of protein per lane) were separated by SDS-PAGE and probed with M1 antibody (A), anti-Gialpha common antibody (B), or anti-Gbeta common antibody (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.

The lack of specific immunoreactive bands recognized by anti-Gialpha common antibody in uninfected Sf9 cells is indicative of the absence of mammalian-type Gi-protein alpha -subunits in Sf9 cell membranes (Fig. 1B). Similar data were obtained by Quehenberger et al. (37), Leopoldt et al. (34), and Grünewald et al. (51). However, in Sf9 membranes from Gialpha 2 baculovirus-infected cells, a strong immunoreactive 40-kDa band appeared, corresponding to the expected molecular mass of Gialpha 2 (31, 52). Of interest, Sf9 membranes express a G-protein beta -subunit which is recognized by the anti-Gbeta common antibody and possesses a molecular mass of 36 kDa (Fig. 1C) (34). In Sf9 cells infected with beta 1gamma 2 baculovirus, an immunoreactive band migrating slightly faster, corresponding to beta 1, appeared, whereas the expression of the insect beta -subunit appeared to decrease. Collectively, our data show that structurally intact FPR, Gialpha 2, and beta 1-subunit can be co-expressed in Sf9 cells. We did not assess the expression of the gamma 2-subunit in Sf9 cells, but the below-described data strongly indicate that functional Gialpha 2beta 1gamma 2 heterotrimers were formed.

The FPR Couples Efficiently to Gialpha 2beta 1gamma 2 but Not to Endogenous G-proteins in Sf9 Membranes-- Quehenberger et al. (37) had shown that the human FPR can be expressed in Sf9 cells but that the GPCR does not couple to the endogenous G-proteins of the insect cells as assessed by the lack of formyl peptide-stimulated GTPase activity and GTPgamma S binding and the lack of high affinity agonist binding. In accordance with these data, basal GTPgamma S binding in Sf9 membranes expressing FPR alone was low, and FMLP increased this GTPgamma S binding only marginally (Fig. 2). The expression of FPR together with Gialpha 2 or beta 1gamma 2 did not induce significant increases in basal GTPgamma S binding and did not result in the appearance of a large stimulatory effect of FMLP. These data clearly show that the FPR cannot form a functional complex with mammalian Gialpha 2 plus insect beta gamma complexes or insect G-protein alpha -subunits plus mammalian beta 1gamma 2 complex. However, the co-expression of FPR together with Gialpha 2beta 1gamma 2 strongly increased basal GTPgamma S binding. FMLP further increased this high GTPgamma S binding, whereas CsH efficiently reduced basal GTPgamma S binding. These data show that the FMLP-liganded FPR can productively activate Gialpha 2beta 1gamma 2. Most intriguingly, the high basal GTPgamma S binding in the complete co-expression system appeared to be largely mediated by the agonist-free FPR since CsH strongly suppressed basal GTPgamma S binding. These data were our first evidence that the FPR expressed in Sf9 membranes is constitutively active and that CsH is an inverse FPR agonist.


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  		Fig. 2.   Reconstitution of FPR ligand-regulated GTPgamma S binding in Sf9 membranes. Membranes from Sf9 cells expressing FPR, FPR plus Gialpha 2, FPR plus beta 1gamma 2, or FPR plus Gialpha 2beta 1gamma 2 were prepared. Reaction mixtures contained Sf9 membranes, 0.4 nM [35S]GTPgamma S, 1 µM GDP, and solvent (basal), FMLP (10 µM) or CsH (3 µM). Incubations were conducted for 45 min. For further details see "Experimental Procedures." Data shown are the means ± S.D. of three experiments.

The inhibitory effect of PTX on basal G-protein activity in HL-60 membranes provided circumstantial evidence for constitutive FPR activity (29-31). Based on these results, we expected PTX also to reduce basal GTPgamma S binding in Sf9 membranes expressing FPR plus Gialpha 2beta 1gamma 2. We treated Sf9 cells with PTX (300 ng/ml) for 24 h before membrane preparation. In HL-60 cells, these conditions are suitable to completely block FMLP-stimulated GTPgamma S binding (31). However, in membranes obtained from PTX-treated Sf9 cells, there was neither a reduction of basal GTPgamma S binding nor FMLP-stimulated GTPgamma S binding compared with control membranes (data not shown). We assume that PTX only poorly penetrates the plasma membrane of Sf9 cells so that the number of non-ADP-ribosylated Gialpha -subunits is still high enough to allow virtually unimpaired coupling of FPR to Gialpha 2beta 1gamma 2. This assumption is supported by the fact that for ADP-ribosylation of endogenous PTX-sensitive G-proteins of Sf9 cells, PTX had to be employed at extremely high concentrations (53). To the best of our knowledge, a successful PTX treatment of intact Sf9 cells expressing a mammalian GPCR and mammalian G-protein has not yet been reported.

Time course of GTPgamma S Binding: FMLP Accelerates While CsH Delays Association of GTPgamma S to Gialpha 2beta 1gamma 2 in Sf9 Membranes-- GTPgamma S binding to G-proteins is quasi-irreversible and follows monophasic saturation kinetics (30). In Sf9 membranes expressing FPR plus Gialpha 2beta 1gamma 2, basal GTPgamma S binding proceeded with a t1/2 of 17 min, and a maximum was reached after ~60 min (Fig. 3). FMLP increased the association rate of GTPgamma S by more than 3-fold (t1/2, 5 min). The relative stimulatory effect of FMLP was most pronounced at early time points (e.g. 145% stimulation at 5 min), whereas at later time points, the stimulatory effect of FMLP became smaller (e.g. only 20% stimulation at 180 min). In contrast to FMLP, CsH delayed the association rate of GTPgamma S by more than 2-fold (t1/2, 36 min), and the inhibitory effect of CsH was pronounced even at late time points of the binding reaction (e.g. 32% inhibition at 180 min versus 50% inhibition at 30 min).


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  		Fig. 3.   Time course of GTPgamma S binding in Sf9 membranes. Membranes from Sf9 cells expressing FPR plus Gialpha 2beta 1gamma 2 were prepared. Sf9 membranes were incubated for the indicated periods in the presence of 1 nM [35S]GTPgamma S plus 4 nM unlabeled GTPgamma S, 1 µM GDP, and solvent (basal), FMLP (10 µM) or CsH (3 µM). For further details see "Experimental Procedures." Data shown are the means ± S.D. of three experiments. Data were best fit to a monophasic saturation hyperbola.

The FPR Is Expressed at Physiologically Relevant Levels in Sf9 Membranes, and the Majority of the FPRs Is Coupled to Gialpha 2beta 1gamma 2-- To address the question of whether the constitutive activity of the FPR in Sf9 cells is only observed because of massive GPCR overexpression that would largely increase the total number of FPRs in the R* state (19, 27), we determined the FPR expression level. Sf9 membranes expressing FPR plus Gialpha 2beta 1gamma 2 bound the radioligand [3H]FMLP in a monophasic and saturable manner (Kd, 3.4 ± 0.8 nM; Bmax, 1.18 ± 0.09 pmol/mg) (Fig. 4A). GTPgamma S uncouples GPCR from G-proteins by activating G-protein alpha -subunits. As a result of this uncoupling, agonist affinity for GPCR is reduced (14, 22, 29, 33). GTPgamma S increased the Kd of [3H]FMLP binding in the Sf9 system to 13.2 ± 4.5 nM and decreased Bmax to 0.34 ± 0.06 pmol/mg. These binding data indicate that ~70% of the expressed FPRs were coupled to Gialpha 2beta 1gamma 2. Of particular importance, the Bmax value of high affinity [3H]FMLP binding in Sf9 membranes was in the same range as in HL-60 cells (1.0-2.9 pmol/mg) (11, 54). Thus, we conclude that the high constitutive activity of the FPR expressed in Sf9 cells cannot be explained by GPCR overexpression.


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  		Fig. 4.   Ligand binding properties of the FLAG epitope- and hexahistidine-tagged FPR expressed in Sf9 membranes. Membranes from Sf9 cells expressing FPR plus Gialpha 2beta 1gamma 2 were prepared. A, Sf9 membranes were incubated in the presence of [3H]FMLP at the concentrations indicated on the abscissa in the absence (basal) or presence of 10 µM GTPgamma S. Nonspecific binding, i.e. the [3H]FMLP binding not competed for by 10 µM unlabeled FMLP, was subtracted from both sets of binding data. For further details, see "Experimental Procedures." Data shown are the means ± S.D. of three experiments. Data were best fit to a monophasic saturation hyperbola. B, Sf9 membranes were incubated in the presence of 5 nM [3H]FMLP and CsH or BocPLPLP at the concentrations indicated on the abscissa. For further details see "Experimental Procedures." Data shown are the means ± S.D. of three experiments. Reactions were conducted for 60 min. Data were best fit to single-site competition curves.

Unaltered Ligand Binding Affinities of the FLAG- and Hexahistidine Tag-modified FPR in Sf9 Membranes-- We introduced an N-terminal FLAG epitope and a C-terminal hexahistidine tag into the FPR (see "Experimental Procedures") to facilitate immunological detection (see Fig. 1A) and future purification (20, 45) of the receptor protein. Therefore, we had to address the question of whether the FLAG epitope- and hexahistidine-tag modifications of our FPR construct alter the ligand binding properties of the GPCR. The Kd value for [3H]FMLP binding to the FLAG epitope- and hexahistidine-tag-modified FPR expressed in Sf9 cells (3.4 ± 0.8 nM) is in close agreement with the Kd value for the unmodified FPR expressed in dibutyryl cAMP-differentiated HL-60 cells (0.7-3.6 nM) (11, 54). We also determined the affinities of BocPLPLP and CsH for the FLAG-epitope- and hexahistidine-tag-modified FPR. BocPLPLP and CsH inhibited [3H]FMLP binding to the receptor according to monophasic functions (Fig. 4B). The Ki value for CsH at the FLAG epitope- and hexahistidine-tag-modified FPR-26 was 287 ± 18 nM, and at the native FPR the Ki value is 100 nM (10, 11). The corresponding values for BocPLPLP at the modified and native FPRs are 1.50 ± 0.19 and 1.46 µM, respectively (10, 11). Taken together, all these data show that the ligand binding properties of the FPR expressed in myeloid cells and those of the FLAG epitope- and hexahistidine-tag-modified FPR expressed in Sf9 cells are very similar. Previous studies with other GPCRs have also shown that the FLAG- and hexahistidine-tag modifications do not interfere with receptor ligand binding properties (45, 46, 55). Additionally, a hexahistidine tag may decrease susceptibility of GPCR to proteolysis (46). Since proteolysis of GPCRs expressed in Sf9 cells can be a serious problem (56), protection against proteolysis by a tag is an important advantage for accurate data analysis. In fact, we did not detect degradation products of the FPR in immunoblots (see Fig. 1A).

GTPgamma S Saturation Binding Studies: a Single FPR Molecule Activates ~7 Gialpha 2beta 1gamma 2 Heterotrimers in Sf9 Membranes-- The observed constitutive activity of the FPR expressed in Sf9 cells could be the result of an abnormal GPCR to G-protein ratio. Specifically, if the number of available G-proteins per FPR molecule in Sf9 membranes were increased relative to physiological conditions, the fraction of receptors in the R* state would increase and, thereby, enhance inverse agonist efficacy (17, 18, 27). By determining the maximum FMLP-stimulated GTPgamma S binding in HL-60 membranes and referring this number to the expression level of G-protein-coupled FPRs, Gierschik et al. (30) and Jacobs et al. (54) estimated that one FPR molecule can activate 4-20 Gi-proteins. At an incubation time of 45 min, i.e. a time suitable to detect both agonist stimulation and inverse agonist inhibition of GTPgamma S binding (see Fig. 3), the Bmax of FMLP-stimulated GTPgamma S binding amounted to 2.83 ± 0.18 pmol/mg, and the Bmax of CsH-inhibited GTPgamma S binding was 3.03 ± 0.22 pmol/mg. Thus, the total concentration of FPR-regulated Gialpha 2beta 1gamma 2 heterotrimers in Sf9 membranes was 5.85 pmol/mg (Fig. 5). If this number is divided by the expression level of G-protein-coupled FPRs (0.84 pmol/mg) (see Fig. 4A), one can estimate that one FPR molecule activates 5-7 Gi-proteins. This number compares favorably to the data obtained in HL-60 membranes (30, 54). About 50% of the FPR-induced G-protein activation in Sf9 membranes is mediated by the agonist-free GPCR, and the remainder is mediated by the agonist-occupied FPR. Taken together, our data show that increased availability of G-proteins in the Sf9 membranes does not explain the observed constitutive activity of the FPR. The Kd value of the FMLP-stimulated GTPgamma S binding was 0.78 ± 0.36 nM, and the Kd value of the CsH-inhibited GTPgamma S binding was not significantly different.


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  		Fig. 5.   Effects of FMLP and CsH on GTPgamma S saturation binding in Sf9 membranes. Membranes from Sf9 cells expressing FPR plus Gialpha 2beta 1gamma 2 were prepared. Sf9 membranes were incubated for 45 min in the presence of 1 nM [35S]GTPgamma S plus unlabeled GTPgamma S at different concentrations to give the final ligand concentrations indicated on the abscissa, 1 µM GDP, and solvent (basal), FMLP (10 µM) or CsH (3 µM). Nonspecific binding was determined in the presence of 10 µM unlabeled GTPgamma S. For each GTPgamma S concentration, the basal GTPgamma S binding was subtracted from GTPgamma S binding observed in the presence of FMLP to calculate the increase in GTPgamma S binding caused by FMLP. From basal GTPgamma S binding values we subtracted GTPgamma S binding observed in the presence of CsH to obtain the decrease of GTPgamma S binding caused by CsH. For further details see "Experimental Procedures." The dotted line is the extrapolation of basal GTPgamma S binding. Data shown are the means ± S.D. of three experiments. Data were best fit to a single-site saturation hyperbola.

Concentration-Response Curves for FMLP, CsH, and BocPLPLP on GTPgamma S Binding in Sf9 Membranes: CsH Is an Inverse FPR Agonist and BocPLPLP Is a Neutral FPR Antagonist-- FMLP stimulated GTPgamma S binding in membranes expressing FPR plus Gialpha 2beta 1gamma 2 with a half-maximal effect at 0.82 ± 0.12 µM and a plateau at 10-100 µM (Fig. 6A). Similar values were obtained for the FMLP-induced Gi-protein activation in HL-60 membranes (31, 54, 57). CsH reduced GTPgamma S binding with a half-maximal effect at 36 ± 19 nM and a plateau at 1-10 µM. Of interest, the potency of CsH at reducing basal GTPgamma S binding was almost 8-fold higher than its Ki value for inhibition of [3H]FMLP binding (see Fig. 4B). In contrast, the potency of FMLP at stimulating GTPgamma S binding was more than 200 times lower than the Kd value for [3H]FMLP binding (see Fig. 4A). These apparent discrepancies could be reconciled when taking into consideration that guanine nucleotides reciprocally alter the affinities of agonist and inverse agonist for GPCR (22, 58) and that ligand competition studies were performed in the absence of guanine nucleotides, whereas GTPgamma S binding studies were performed in the presence of guanine nucleotides (GTPgamma S and GDP) (see "Experimental Procedures"). In particular, GDP and GTPgamma S both reduce the affinity of FPR for agonist (59), whereas guanine nucleotides increase the affinity of GPCR for inverse agonist, presumably by facilitating stabilization of GPCR in the G-protein-uncoupled R state (22, 58).


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  		Fig. 6.   Concentration-response curves for FMLP, CsH, and BocPLPLP on GTPgamma S binding in Sf9 membranes. Membranes from Sf9 cells expressing FPR plus Gialpha 2beta 1gamma 2 were prepared. A, Sf9 membranes were incubated for 45 min in the presence of 0.4 nM [35S]GTPgamma S, 1 µM GDP, and FPR ligands at the concentrations indicated on the abscissa. B, Sf9 membranes were incubated for 45 min in the presence of 0.4 nM [35S]GTPgamma S, 1 µM GDP, and CsH at the concentrations indicated on the abscissa. Reaction mixtures additionally contained solvent (control) or BocPLPLP (3 µM). For further details see "Experimental Procedures." Data shown are the means ± S.D. of three experiments. Data shown in B were best fit to single-site competition curves.

In contrast to CsH, BocPLPLP at concentrations up to 3 µM, e.g. a concentration twice as high as its Ki value for binding to the FPR (see Fig. 4B), had no inhibitory effect on basal GTPgamma S binding (Fig. 6A). At a concentration of 10 µM, BocPLPLP had a small inhibitory effect on GTPgamma S binding. However, if BocPLPLP, by analogy to CsH, had been an inverse FPR agonist, the peptide would have been expected to reduce GTPgamma S binding with a potency that is higher than its Ki value for binding to the FPR. Most likely, the small inhibition of basal GTPgamma S binding by BocPLPLP at 10 µM represents a nonspecific effect of this extremely hydrophobic peptide on Gi-proteins rather than a specific FPR-mediated effect. Indeed, several hydrophobic peptides have been shown to inhibit the function of PTX-sensitive G-proteins directly (60).

According to the two-state model of GPCR activation, neutral antagonists do not only block the effects of agonists but also the effects of inverse agonists (18, 22, 23). Because BocPLPLP at concentrations of up to 3 µM did not affect basal GTPgamma S binding, we could study the effect of the antagonist on CsH-mediated inhibition of GTPgamma S binding. In fact, BocPLPLP at 3 µM shifted the concentration-response curve for CsH on GTPgamma S binding by 3-fold to the right (Fig. 6B). As reported previously for human neutrophils and dibutyryl cAMP-differentiated HL-60 cells (10, 11), BocPLPLP also competitively antagonized the stimulatory effect of FMLP in the Sf9 cell system (data not shown).

Regulation of GTPgamma S Binding in Sf9 Membranes by NaCl and KCl: Na+ Stabilizes the FPR in the R State and Thereby Reduces the Inverse Agonistic Efficacy of CsH-- NaCl efficiently reduces the basal G-protein activity in membranes expressing constitutively active Gi-protein-linked GPCRs (26, 32). As the result of this reduction in basal G-protein activity by NaCl, the efficacy of inverse agonist is reduced (26, 32). Of interest, NaCl also efficiently reduces the basal G-protein activity in myeloid membranes expressing FPR (29, 30). These findings prompted us to analyze the effects of NaCl on GTPgamma S binding in Sf9 membranes expressing FPR plus Gialpha 2beta 1gamma 2. NaCl reduced basal GTPgamma S binding to a value that corresponds to maximum CsH-inhibited GTPgamma S binding, i.e. NaCl reduced the inverse agonistic efficacy of CsH (Fig. 7A). In contrast, NaCl increased the absolute and relative stimulatory effect of FMLP on GTPgamma S binding. Note that KCl at concentration as high as 150 mM could not abolish the inhibitory effect of CsH on GTPgamma S binding (Fig. 7B).


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  		Fig. 7.   Effects of NaCl and KCl on GTPgamma S binding in Sf9 membranes. Membranes from Sf9 expressing FPR plus Gialpha 2beta 1gamma 2 were prepared. A, Sf9 membranes were incubated for 45 min in the presence of 0.4 nM [35S]GTPgamma S, 1 µM GDP, and NaCl at the concentrations indicated on the abscissa. Reaction mixtures additionally contained solvent (basal), FMLP (10 µM), or CsH (3 µM). B, Sf9 membranes were incubated for 45 min in the presence of 0.4 nM [35S]GTPgamma S, 1 µM GDP, and KCl at the concentrations indicated on the abscissa. Reaction mixtures additionally contained solvent (basal), FMLP (10 µM), or CsH (3 µM). For further details see "Experimental Procedures." Data shown are the means ± S.D. of three experiments.

The differential effects of NaCl and KCl on basal GTPgamma S binding in Sf9 membranes clearly show that the potent inhibition caused by NaCl is not due to an increase in ionic strength of the incubation medium or due to Cl- anions but rather due to Na+ cations. The fact that Na+ abolished the inhibitory effect of CsH on GTPgamma S binding while leaving intact the total FPR ligand-regulated GTPgamma S binding, i.e. the difference between maximum CsH-inhibited and maximum FMLP-stimulated GTPgamma S binding, suggests that both Na+ and CsH stabilize the FPR in the R state. Considering mutational studies with other GPCRs (61-63), it is likely that the highly conserved Asp-71 in the second transmembrane domain of the FPR (64) represents an important molecular target of Na+.

Of interest, the inhibitory effect of NaCl on basal GTPgamma S binding was half-maximal at a concentration as low as 5 mM and reached a maximum already at 20-50 mM. In this context, it should be noted that the intracellular free Na+ concentration in unstimulated human neutrophils is ~10 mM and rises up to ~20 mM upon stimulation with FMLP (65). The similarities of the intracellular Na+ concentration with the Na+ concentrations that regulate FPR activity in vitro raise the question inasmuch as Na+ influx could provide a signal by which the constitutive activity of the FPR is decreased. However, it is unknown whether in vivo, Na+ has free access to the intramembranously buried Asp-71 of the FPR. It is also unknown whether Na+ reaches Asp-71 from the intracellular space, from the extracellular space, or from both compartments.

Studies with HEK293 Membranes-- The glycosylation pattern of the FPR expressed in mammalian and insect cells is quite different (Fig. 1A) (37, 50). To address the question of whether the glycosylation pattern of the FPR or the cellular background critically determine constitutive GPCR activity, we also expressed the FPR in HEK293 cells. This mammalian expression system is suitable for reconstituting Gi-protein coupling of chemoattractant receptors (38, 39). For our studies, we chose a cell clone that stably expressed FPR with a Bmax of 1.11 pmol/mg, i.e. a level comparable to the FPR expression level in Sf9 cells and HL-60 cells (Fig. 4A) (11, 54). The Kd value of the monophasic [3H]FMLP saturation binding curve in HEK293 membranes was 0.75 ± 0.20 nM and thus comparable to the Kd values observed for Sf9 membranes and (Fig. 4A) (11, 54).

To study the G-protein coupling of the FPR expressed in HEK293 membranes, we performed GTPgamma S binding studies (Table I). In the absence of NaCl, FMLP did not exhibit a stimulatory effect on GTPgamma S binding, whereas CsH reduced basal GTPgamma S binding by about 20%. NaCl (100 mM) reduced basal GTPgamma S binding in HEK293 membranes by almost 50% and unmasked a robust stimulatory effect of FMLP on GTPgamma S binding. In contrast, NaCl abolished the inhibitory effect of CsH on GTPgamma S binding in HEK293 membranes. Overall, the ligand and Na+ regulation of GTPgamma S binding by FPR in HEK293 membranes is similar to the regulation observed in Sf9 membranes. Of interest, the apparent constitutive activity of the FPR expressed in HEK293 cells is even higher than in Sf9 membranes since FMLP failed to stimulate GTPgamma S binding in HEK293 membranes in the absence of NaCl. However, it should be noted that the absolute ligand-regulated GTPgamma S binding in HEK293 membranes is substantially lower than in Sf9 membranes (compare Fig. 7 with Table I, for example). In addition, the relative inhibitory effect of CsH on GTPgamma S binding in HEK293 membranes was considerably smaller than in Sf9 membranes (~20% inhibition versus ~50% inhibition). This difference between the two expression systems may indicate that in HEK293 cells, the concentration of G-proteins that can couple to the FPR is lower than in Sf9 cells. From a practical point of view, our data clearly show that the Sf9 system is more sensitive than the HEK293 system for analysis of constitutive FPR activity on the membrane level. However, HEK293 membranes are still more sensitive in this respect than dibutyryl cAMP-differentiated HL-60 membranes since, in the latter system, inverse agonistic activity of CsH could not be detected at all (11).

                              
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Table I
Regulation of GTPgamma S binding by FMLP, CsH, and Na+ in HEK293 membranes
Membranes from HEK293 cells stably expressing FPR were prepared. HEK293 membranes were incubated for 45 min in the presence of 0.4 nM [35S]GTPgamma S, 1 µM GDP, solvent (basal), FMLP, and CsH at the indicated concentrations. Reaction mixtures additionally contained distilled water (control) or NaCl (100mM). For further experimental details, see "Experimental Procedures." Data shown are the means ± S.D. of three experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

High Constitutive Activity of the Human FPR: Implications for Other Chemoattractant Receptors and Structural Basis-- Our data can be interpreted in the framework of the two-state model of GPCR activation (14-17, 20, 21). Specifically, the FPR isomerizes from an inactive state R to an active R* state, and this isomerization occurs at a substantial rate even in the absence of agonist. The spontaneous R- to R* transition results in high basal GTPgamma S binding (Fig. 8A). FMLP stabilizes the FPR in the R* state and, thereby, increases basal GTPgamma S binding to Gialpha 2beta 1gamma 2 in Sf9 membranes (Fig. 8B), whereas CsH and Na+ stabilize the FPR in the R state and reduce the high basal GTPgamma S binding (Fig. 8C). The neutral antagonist BocPLPLP inhibits both the effects of agonist (Fig. 8B) and inverse agonist (Fig. 8C). Taken together, the FPR is the first member of the chemoattractant receptor family (4, 66) unequivocally identified as being constitutively active.


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  		Fig. 8.   Constitutive activity of the FPR. A, based on studies with Sf9 cells and HEK293 cells expressing FPR, studies with FPR/C5a receptor chimeras and studies with intact neutrophils and HL-60 cells as well as HL-60 membranes, the following simple model of FPR activation can be developed. The FPR isomerizes from the inactive state R to the active state R* in an agonist-independent manner and increases GTPgamma S binding to Gi-proteins such as Gialpha 2beta 1gamma 2. B, the agonist FMLP stabilizes the R* state of the FPR and increases basal GTPgamma S binding to Gi-proteins. The neutral FPR antagonist BocPLPLP blocks the stimulatory effects of FMLP. C, Na+ and the inverse agonist CsH stabilize the FPR in the inactive R state and, thereby, reduce the basal GTPgamma S binding to Gi-proteins. BocPLPLP antagonizes the inhibitory effects of CsH. For further details see text.

We can exclude the possibility that the constitutive activity of the FPR is attributable to receptor overexpression or excessive availability of G-proteins (Figs. 4A and 5 and Table I). Moreover, we observed constitutive FPR activity in a mammalian and an insect expression system, excluding the possibility that differences in cellular background or glycosylation have a profound impact on constitutive GPCR activity. Thus, it appears that the constitutive activity of the FPR observed in two unrelated and very different expression systems reflects an authentic property of this GPCR.

Indeed, constitutive activity has been described for numerous wild-type GPCRs including biogenic amine receptors (e.g. beta 2, hydroxytryptamine2C, and histamine H2 receptors) (17, 19-23, 36), neuropeptide (delta -opioid) receptors (24, 32, 67), muscarinic (28, 68), and thyrotropin receptors (25). Of interest, the constitutive activity of individual GPCRs differs substantially from each other. For example, the thyrotropin receptor has a much higher constitutive activity than the luteinizing/chorionic gonadotropin receptor, although the two GPCRs are highly homologous (25). Given the fact that at a physiologically relevant receptor expression level and receptor to G-protein stoichiometry (Figs. 4A and 5 and Table I), the maximum effect of inverse agonist was of similar magnitude as, or even bigger than, the maximum effect of agonist, the FPR can certainly be classified as a highly constitutively active GPCR. Considering that the FPR shows substantial homology to the complement C5a, the platelet-activating factor, and leukotriene B4 receptors (4, 69-71), it is likely that these chemoattractant receptors possess at least some constitutive activity as well.

Our data raise the question of what the structural basis of the constitutive FPR activity may be. So far, a common structural motif in GPCRs resulting in constitutive activity has not yet been identified. It rather appears than in different GPCR families, different receptor domains are of importance for constraining GPCR in an inactive and agonist-activable conformation (72). Based on the Na+ sensitivity of the constitutive FPR activity and Na+ sensitivity data obtained for other GPCRs (61-64), Asp-71 in the second transmembrane domain of the FPR may play a role in regulating the R to R* transition of this GPCR. Of particular interest, a chimeric FPR in which the first intracellular loop of the FPR is replaced by the corresponding domain of the C5a receptor possesses a higher constitutive activity than wild-type FPR (73). These findings point to the importance of the first cytosolic loop of the FPR in constraining this GPCR in an inactive conformation and support our suggestion that other members of the chemoattractant receptor family may also be constitutively active, perhaps even more constitutively active than the FPR. Since the first cytosolic loop of the FPR is important for receptor/G-protein coupling (74), it is conceivable that structural changes in this region of the receptor molecule affect the ability of the G-protein to interact with GPCR and, consequently, to stabilize the R* state. Thus, the first cytosolic loop of the C5a receptor may be more efficient at promoting receptor/G-protein coupling than than the corresponding FPR domain so that G-protein can more efficiently stabilize the R* state and, thereby, increase the apparent constitutive GPCR activity (18, 33). In this context, it is important to note that the first cytosolic loop is also involved in constitutive activation of the melanocyte-stimulating hormone receptor (75) and the parathyroid hormone-parathyroid hormone-related peptide receptor (76). The third transmembrane domain of the FPR may also be of importance for determining constitutive GPCR activity (see discussion below).

Possible Biological Roles of Constitutive FPR Activity-- CsH is a naturally occurring inverse agonist. This raises the question of whether these compounds could have any biological function. CsH is a metabolite of the fungus imperfectus T. inflatum Gams which resides in the soil of Wisconsin and the Hardanger Vidda (Norway) (13). Because of its structural similarity to cyclosporin A, CsH is likely to be absorbed in the gastrointestinal tract after oral ingestion (11). Thus, CsH could reach the systemic circulation of mammals feeding on roots, worms, and insects present in the soil and exert biological effects on neutrophils.

Evidence for the relevance of constitutive activity of wild-type GPCRs in intact cells has already been provided for the 5-hydroxytryptamine2C receptor (22), beta -adrenergic receptors (77, 78), muscarinic receptors (79), and alpha 1-adrenoreceptors (80). Several pieces of evidence point to the relevance of constitutive FPR activity in intact cells. First, in intact myeloid cells, inhibitory effects of PTX on "basal" activities have been repeatedly observed. Specifically, PTX reduces basal inositol phosphate production in human neutrophils and HL-60 cells (81, 82), Na+-dependent uridine uptake in HL-60 cells (83), and basal Na+ entry in rabbit neutrophils (84). It should also be mentioned that PTX can inhibit apparently agonist-independent long term events in HL-60 cells such as Me2SO-induced differentiation (85). Second, the expression level of FPR is increased by mediators of inflammation such as interferon-gamma and tumor necrosis factor-alpha (86, 87). Increased FPR expression increases the absolute number of FPRs in the R* state and, thereby, could reduce the treshold concentration of FMLP at which neutrophils generate reactive oxygen species and release lysosomal enzymes ("priming") (3). Third, elevated basal phospholipase C activity has been observed for the FPR co-expressed with the PTX-insensitive G-protein Galpha 16 in COS-7 cells (73).

Why Can the Inverse Agonistic Activity of CsH at the FPR Not be Detected in HL-60 Membranes?-- Dibutyryl cAMP-differentiated HL-60 cells are a very sensitive model system for studying FPR/Gi-protein interaction (7). Nonetheless, in our previous study (11) we failed to uncover an inhibitory effect of CsH on basal Gi-protein activity in this system, and we concluded that CsH is a neutral FPR antagonist. Our present data show that CsH must be classified as an inverse agonist. We can offer two explanations which alone or together could to reconcile the divergent effects of CsH in our previous and our present study.

First, dibutyryl cAMP-differentiated HL-60 cells express two different FPR subtypes, referred to as FPR-26 and FPR-98, respectively (40). In our present study, we analyzed FPR-26 (see "Experimental Procedures"). The precise ratio of FPR-26 and FPR-96 in HL-60 cells is unknown. FPR-26 and FPR-98 differ from each other in two amino acids, namely at positions 101 and 346 (40). These amino acid positions are located in the third transmembrane domain of the receptor and the receptor C terminus, respectively. Both receptor regions are presumably not critical for G-protein coupling (74). However, considering the fact that mutations in the third transmembrane domain of certain GPCRs, i.e. in alpha 1-adrenergic and thyrotropin receptors (72), can lead to increases in constitutive activity, it cannot be excluded that FPR-26 and FPR-98 exhibit different degrees of constitutive activity. An implication of any difference in constitutive activity of FPR isoforms would be that dibutyryl cAMP-differentiated HL-60 cells predominantly express an FPR isoform with low constitutive activity. Based on our current results, this should be the FPR-98.

Second, dibutyryl cAMP-differentiated HL-60 cells express at least eight different types of Gi-protein-linked GPCRs (FPR-26, FPR-98, P2U receptors, histamine H1 receptors, and receptors for C5a, platelet-activating factor, leukotriene B4, and sphingosylphosphorylcholine) (7, 40, 88) all of which may exhibit some degree of constitutive activity. In fact, studies with FPR/C5a receptor chimeras support the view that not only FPRs but also other chemoattractant receptors are constitutively active (73). PTX reduces the basal Gi-protein activity in membranes of dibutyryl cAMP-differentiated HL-60 cells by 20-40% (31, 57). Thus, the relative contribution of FPRs to the total basal Gi-protein activity supported by all constitutively active GPCR types in HL-60 membranes may be too small and beyond the sensitivity limit of the available G-protein assays to detect an effect of CsH. In contrast to HL-60 membranes, Sf9 membranes and HEK293 membranes express a single defined GPCR subtype, thereby eliminating the contribution of other GPCRs on basal G-protein activity and greatly increasing the assay sensitivity.

In conclusion, we have shown that the human FPR expressed in Sf9 cells and HEK293 cells possesses high constitutive activity. CsH is an inverse FPR agonist and, like Na+, stabilizes the FPR in the inactive R state. Constitutive FPR activity is probably of importance in vivo.

    ACKNOWLEDGEMENTS

We are most indebted to Dr. Brian K. Kobilka for continuous support and helpful suggestions; to Dr. F. Boulay for providing FPR-26 cDNA; to Dr. A. G. Gilman for providing the Gialpha 2 baculovirus; to Dr. P. Gierschik for donating beta 1gamma 2 baculovirus; and Drs. B. Nürnberg and G. Schultz for providing anti-Gialpha common and anti-Gbeta common antibodies. The generous gift of CsH from Novartis (Basel, Switzerland) is appreciated. We are also most grateful to Dr. T. W. Lee for skilled advice with the immunoblots. Finally, we would like to thank the Reviewer of this paper for the constructive criticism and many helpful suggestions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Pharmacology and Toxicology, The University of Kansas, 5064 Malott Hall, Lawrence, KS 66045.

§ Recipient of research fellowship of the Deutsche Forschungsgemeinschaft.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, The University of Kansas, 5064 Malott Hall, Lawrence, KS 66045. Tel.: 785-864-3538; Fax: 785-864-5219.

parallel Supported by National Institutes of Health Medical Scientist Training Program GM 07365 from the NIGMS.

The abbreviations used are: FMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanineBocPLPLP, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L-phenylalanineCsH, cyclosporin HFPR, formyl peptide receptorGPCR, G-protein-coupled receptorGTPgamma S, guanosine 5'-O-(3-thiotriphosphate)PTX, pertussis toxinSf9 cells, Spodoptera frugiperda cellsbp, base pair(s)PCR, polymerase chain reactionPAGE, polyacrylamide gel electrophoresis.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Rossi, F. (1986) Biochim. Biophys. Acta 853, 65-89[Medline] [Order article via Infotrieve]
  2. Sandborg, R. R., and Smolen, J. E. (1988) Lab. Invest. 59, 300-320[Medline] [Order article via Infotrieve]
  3. Seifert, R., and Schultz, G. (1991) Rev. Physiol. Biochem. Pharmacol. 117, 1-338[Medline] [Order article via Infotrieve]
  4. Murphy, P. M. (1994) Annu. Rev. Immunol. 12, 593-633[CrossRef][Medline] [Order article via Infotrieve]
  5. Kobilka, B. K. (1992) Annu. Rev. Neurosci. 15, 87-114[CrossRef][Medline] [Order article via Infotrieve]
  6. Dillon, S. B., Verghese, M. W., and Snyderman, R. (1988) Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 55, 65-80[Medline] [Order article via Infotrieve]
  7. Klinker, J. F., Wenzel-Seifert, K., and Seifert, R. (1996) Gen. Pharmacol. 27, 33-54[CrossRef][Medline] [Order article via Infotrieve]
  8. de Paulis, A., Ciccarelli, A., de Crescenzo, G., Cirillo, R., Patella, V., and Marone, G. (1996) J. Allergy Clin. Immunol. 98, 152-164[CrossRef][Medline] [Order article via Infotrieve]
  9. Kanaho, Y., Kermode, J. C., and Becker, E. L. (1990) J. Leukocyte Biol. 47, 420-428[Abstract]
  10. Wenzel-Seifert, K., Grünbaum, L., and Seifert, R. (1991) J. Immunol. 147, 1940-1946[Abstract]
  11. Wenzel-Seifert, K., and Seifert, R. (1993) J. Immunol. 150, 4591-4599[Abstract]
  12. Traber, R., Loosli, H.-R., Hofmann, H., Kuhn, M., and von Wartburg, A. (1982) Helv. Chim. Acta 65, 1655-1677[CrossRef]
  13. von Wartburg, A., and Traber, R. (1986) Prog. Allergy 38, 28-45[Medline] [Order article via Infotrieve]
  14. Lefkowitz, R. J., Cotecchia, S., Samama, P., and Costa, T. (1993) Trends Pharmacol. Sci. 14, 303-307[CrossRef][Medline] [Order article via Infotrieve]
  15. Leff, P. (1995) Trends Pharmacol. Sci. 16, 89-97[CrossRef][Medline] [Order article via Infotrieve]
  16. Schütz, W., and Freissmuth, M. (1992) Trends Pharmacol. Sci. 13, 376-380[CrossRef][Medline] [Order article via Infotrieve]
  17. Seifert, R., Lee, T. W., Lam, V. T., and Kobilka, B. K. (1998) Eur. J. Biochem. 255, 369-382[Medline] [Order article via Infotrieve]
  18. Burstein, E. S., Spalding, T. A., and Brann, M. R. (1997) Mol. Pharmacol. 51, 312-319[Abstract/Free Full Text]
  19. Bond, R. A., Leff, P., Johnson, T. D., Milano, C. A., Rockman, H. A., McMinn, T. R., Apparsundaram, S., Hyek, M. F., Kenakin, T. P., Allen, L. F., and Lefkowitz, R. J. (1995) Nature 374, 272-276[CrossRef][Medline] [Order article via Infotrieve]
  20. Gether, U., Lin, S., and Kobilka, B. K. (1995) J. Biol. Chem. 270, 28268-28275[Abstract/Free Full Text]
  21. Chidiac, P., Hebert, T. E., Valiquette, M., Dennis, M., and Bouvier, M. (1994) Mol. Pharmacol. 45, 490-499[Abstract]
  22. Barker, E. L., Westphal, R. S., Schmidt, D., and Sanders-Bush, E. (1994) J. Biol. Chem. 269, 11687-11690[Abstract/Free Full Text]
  23. Smit, M. J., Leurs, R., Alewijnse, A. E., Blauw, J., Van Nieuw Amerongen, G. P., Van De Vrede, Y., Roovers, E., and Timmerman, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6802-6807[Abstract/Free Full Text]
  24. Costa, T., and Herz, A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7321-7325[Abstract/Free Full Text]
  25. Cetani, F., Tonacchera, M., and Vassart, G. (1996) FEBS Lett. 378, 27-31[CrossRef][Medline] [Order article via Infotrieve]
  26. Tian, W. N., Duzic, E., Lanier, S. M., and Deth, R. C. (1994) Mol. Pharmacol. 45, 524-531[Abstract]
  27. Kenakin, T. (1997) Trends Pharmacol. Sci. 18, 456-464[Medline] [Order article via Infotrieve]
  28. Jakubik, J., Bacakova, L., el-Fakahany, E. E., and Tucek, S. (1995) FEBS Lett. 377, 275-279[CrossRef][Medline] [Order article via Infotrieve]
  29. Gierschik, P., Sidiropoulos, D., Steisslinger, M., and Jakobs, K. H. (1989) Eur. J. Pharmacol. 172, 481-492[CrossRef][Medline] [Order article via Infotrieve]
  30. Gierschik, P., Moghtader, R., Straub, C., Dieterich, K., and Jakobs, K. H. (1991) Eur. J. Biochem. 197, 725-732[Medline] [Order article via Infotrieve]
  31. Klinker, J. F., Schwaner, I., Offermanns, S., Hagelüken, A., and Seifert, R. (1994) Biochem. Pharmacol. 48, 1857-1864[CrossRef][Medline] [Order article via Infotrieve]
  32. Costa, T., Lang, J., Gless, C., and Herz, A. (1990) Mol. Pharmacol. 37, 383-394[Abstract]
  33. Seifert, R., Wenzel-Seifert, K., Lee, T. W., Gether, U., Sanders-Bush, E., and Kobilka, B. K. (1998) J. Biol. Chem. 273, 5109-5116[Abstract/Free Full Text]
  34. Leopoldt, D., Harteneck, C., and Nürnberg, B. (1997) Naunyn-Schmiedeberg's Arch. Pharmacol. 356, 216-224[CrossRef][Medline] [Order article via Infotrieve]
  35. Barr, A. J., and Manning, D. R. (1997) J. Biol. Chem. 272, 32979-32987[Abstract/Free Full Text]
  36. Hartman, J. I., IV, and Northup, J. K. (1996) J. Biol. Chem. 271, 22591-22597[Abstract/Free Full Text]
  37. Quehenberger, O., Prossnitz, E. R., Cochrane, C. G., and Ye, R. D. (1992) J. Biol. Chem. 267, 19757-19760[Abstract/Free Full Text]
  38. Buhl, A. M., Osawa, S., and Johnson, G. L. (1995) J. Biol. Chem. 270, 19828-19832[Abstract/Free Full Text]
  39. Neptune, E. R., and Bourne, H. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14489-14494[Abstract/Free Full Text]
  40. Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochemistry 29, 11123-11133[CrossRef][Medline] [Order article via Infotrieve]
  41. Dietrich, A., Meister, M., Spicher, K., Schultz, G., Camps, M., and Gierschik, P. (1992) FEBS Lett. 313, 220-224[CrossRef][Medline] [Order article via Infotrieve]
  42. Hinsch, K. D., Tychowiecka, I., Gausepohl, H., Frank, R., Rosenthal, W., and Schultz, G. (1989) Biochim. Biophys. Acta 1013, 60-67[Medline] [Order article via Infotrieve]
  43. Singer, S. J. (1990) Annu. Rev. Cell Biol. 6, 247-296[CrossRef]
  44. Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol. Chem. 267, 21995-21998[Abstract/Free Full Text]
  45. Kobilka, B. K. (1995) Anal. Biochem. 231, 269-271[CrossRef][Medline] [Order article via Infotrieve]
  46. Gimpl, G., Anders, J., Thiele, C., and Fahrenholz, F. (1996) Eur. J. Biochem. 237, 768-777[Medline] [Order article via Infotrieve]
  47. von Zastrow, M., and Kobilka, B. K. (1992) J. Biol. Chem. 267, 3530-3538[Abstract/Free Full Text]
  48. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  49. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract/Free Full Text]
  50. Cupo, J. F., Allen, R. A., Jesaitis, A. J., and Bokoch, G. M. (1989) Biochim. Biophys. Acta 982, 31-40[Medline] [Order article via Infotrieve]
  51. Grünewald, S., Reiländer, H., and Michel, H. (1996) Biochemistry 35, 15162-15173[CrossRef][Medline] [Order article via Infotrieve]
  52. Gierschik, P., Sidiropoulos, D., and Jakobs, K. H. (1989) J. Biol. Chem. 264, 21470-21473[Abstract/Free Full Text]
  53. Ng, G. Y., George, S. R., Zastawny, R. L., Caron, M., Bouvier, M., Dennis, M., and O'Dowd, B. F. (1993) Biochemistry 32, 11727-11733[CrossRef][Medline] [Order article via Infotrieve]
  54. Jacobs, A. A., Huber, J. L., Ward, R. A., Klein, J. B., and McLeish, K. R. (1995) J. Leukocyte Biol. 57, 679-686[Abstract]
  55. Robeva, A. S., Woodard, R., Luthin, D. R., Taylor, H. E., and Linden, J. (1996) Biochem. Pharmacol. 51, 545-555[CrossRef][Medline] [Order article via Infotrieve]
  56. Weill, C., Autelitano, F., Guenet, C., Heitz, F., Goeldner, M., and Ilien, B. (1997) Eur. J. Pharmacol. 333, 269-278[CrossRef][Medline] [Order article via Infotrieve]
  57. Klinker, J. F., Hagelüken, A., Grünbaum, L., Heilmann, I., Nürnberg, B., Harhammer, R., Offermanns, S., Schwaner, I., Ervens, J., Wenzel-Seifert, K., Müller, T., and Seifert, R. (1994) Biochem. J. 304, 377-383
  58. Leeb-Lundberg, L. M., Mathis, S. A., and Herzig, M. C. (1994) J. Biol. Chem. 269, 25970-25973[Abstract/Free Full Text]
  59. Gierschik, P., Steisslinger, M., Sidiropoulos, D., Herrmann, E., and Jakobs, K. H. (1989) Eur. J. Biochem. 183, 97-105[Medline] [Order article via Infotrieve]
  60. Mukai, H., Munekata, E., and Higashijima, T. (1992) J. Biol. Chem. 267, 16237-16243[Abstract/Free Full Text]
  61. Ceresa, B. P., and Limbird, L. E. (1994) J. Biol. Chem. 269, 29557-29564[Abstract/Free Full Text]
  62. Kong, H., Raynor, K., Yasuda, K., Bell, G. I., and Reisine, T. (1993) Mol. Pharmacol. 44, 380-384[Abstract]
  63. Neve, K. A., Cox, B. A., Henningsen, R. A., Spanoyannis, A., and Neve, R. L. (1991) Mol. Pharmacol. 39, 733-739[Abstract]
  64. Prossnitz, E. R., Schreiber, R. E., Bokoch, G. M., and Ye, R. D. (1995) J. Biol. Chem. 270, 10686-10694[Abstract/Free Full Text]
  65. Wenzel-Seifert, K., Krautwurst, D., Musgrave, I., and Seifert, R. (1996) Biochem. J. 314, 679-686
  66. Gerard, C., and Gerard, N. P. (1994) Annu. Rev. Immunol. 12, 775-808[CrossRef][Medline] [Order article via Infotrieve]
  67. Campbell, J. J., Qin, S., Bacon, K. B., Mackay, C. R., and Butcher, E. C. (1996) J. Cell Biol. 134, 255-266[Abstract/Free Full Text]
  68. Hilf, G., and Jakobs, K. H. (1992) Eur. J. Pharmacol. 225, 245-252[CrossRef][Medline] [Order article via Infotrieve]
  69. Gerard, N. P., and Gerard, C. (1991) Nature 349, 614-617[CrossRef][Medline] [Order article via Infotrieve]
  70. Ye, R. D., Prossnitz, E. R., Zou, A. H., and Cochrane, C. G. (1991) Biochem. Biophys. Res. Commun. 180, 105-111[CrossRef][Medline] [Order article via Infotrieve]
  71. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624[CrossRef][Medline] [Order article via Infotrieve]
  72. Scheer, A., and Cotecchia, S. (1997) J. Recept. Signal Transduct. Res. 17, 57-73[Medline] [Order article via Infotrieve]
  73. Amatruda, T. T., III, Dragas-Graonic, S., Holmes, R., and Perez, H. D. (1995) J. Biol. Chem. 270, 28010-28013[Abstract/Free Full Text]
  74. Bommakanti, R. K., Dratz, E. A., Siemsen, D. W., and Jesaitis, A. J. (1995) Biochemistry 34, 6720-6728[CrossRef][Medline] [Order article via Infotrieve]
  75. Robbins, L. S., Nadeau, J. H., Johnson, K. R., Kelly, M. A., Roselli-Rehfuss, L., Baack, E., Mountjoy, K. G., and Cone, R. D. (1993) Cell 72, 827-834[CrossRef][Medline] [Order article via Infotrieve]
  76. Schipani, E., Kruse, K., and Jüppner, H. (1995) Science 268, 98-100[Abstract/Free Full Text]
  77. Götze, K., and Jakobs, K. H. (1994) Eur. J. Pharmacol. 268, 151-158[CrossRef][Medline] [Order article via Infotrieve]
  78. Mewes, T., Dutz, S., Ravens, U., and Jakobs, K. H. (1993) Circulation 88, 2916-2922[Abstract/Free Full Text]
  79. Hanf, R., Li, Y., Szabo, G., and Fischmeister, R. (1993) J. Physiol. (Lond.) 461, 743-765[Abstract/Free Full Text]
  80. Noguera, M. A., Ivorra, M. D., and D'Ocon, P. (1996) Br. J. Pharmacol 119, 158-164[Medline] [Order article via Infotrieve]
  81. Krause, K. H., Schlegel, W., Wollheim, C. B., Andersson, T., Waldvogel, F. A., and Lew, P. D. (1985) J. Clin. Invest. 76, 1348-1354
  82. Brandt, S. J., Dougherty, R. W., Lapetina, E. G., and Niedel, J. E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3277-3280[Abstract/Free Full Text]
  83. Sokoloski, J. A., Sartorelli, A. C., Handschumacher, R. E., and Lee, C. W. (1991) Biochem. J. 280, 515-519
  84. Volpi, M., Naccache, P. H., Molski, T. F., Shefcyk, J., Huang, C. K., Marsh, M. L., Munoz, J., Becker, E. L., and Sha'afi, R. I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2708-2712[Abstract/Free Full Text]
  85. Naccache, P. H., Caon, A. C., Gilbert, C., Chouinard, G., and McColl, S. R. (1991) Blood 78, 2534-2541[Abstract/Free Full Text]
  86. Klein, J. B., Scherzer, J. A., and McLeish, K. R. (1992) J. Immunol. 148, 2483-2488[Abstract]
  87. McLeish, K. R., Klein, J. B., Schepers, T., and Sonnenfeld, G. (1991) Biochem. J. 279, 455-460
  88. Van Koppen, C. J., Meyer Zu Heringdorf, D., Zhang, C., Laser, K. T., and Jakobs, K. H. (1996) Mol. Pharmacol. 49, 956-961[Abstract]

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