High Constitutive Activity of the Human Formyl Peptide Receptor

Abstract

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 Giα2β1γ2reconstitutedN-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-stimulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding and GTPγS-sensitive high affinity [3H]FMLP binding. The FPR “antagonist” cyclosporin H (CsH) potently and efficiently reduced basal GTPγS binding in Sf9 membranes. Another FPR antagonist,N-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine did not inhibit basal GTPγS binding but blocked the inhibitory effect of CsH on GTPγS binding. Na+ reduced basal GTPγS 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. 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 Giα2β1γ2reconstitutedN-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-stimulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding and GTPγS-sensitive high affinity [3H]FMLP binding. The FPR “antagonist” cyclosporin H (CsH) potently and efficiently reduced basal GTPγS binding in Sf9 membranes. Another FPR antagonist,N-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine did not inhibit basal GTPγS binding but blocked the inhibitory effect of CsH on GTPγS binding. Na+ reduced basal GTPγS 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. N-formyl-l-methionyl-l-leucyl-l-phenylalanine N-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine cyclosporin H formyl peptide receptor G-protein-coupled receptor guanosine 5′-O-(3-thiotriphosphate) pertussis toxin Spodoptera frugiperda cells base pair(s) polymerase chain reaction polyacrylamide gel electrophoresis. Human neutrophils play a key role in host defense against bacterial infections (1Rossi F. Biochim. Biophys. Acta. 1986; 853: 65-89Crossref PubMed Scopus (582) Google Scholar, 2Sandborg R.R. Smolen J.E. Lab. Invest. 1988; 59: 300-320PubMed Google Scholar, 3Seifert R. Schultz G. Rev. Physiol. Biochem. Pharmacol. 1991; 117: 1-338PubMed Google Scholar). Neutrophils are activated by the bacterial formyl peptideN-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 (1Rossi F. Biochim. Biophys. Acta. 1986; 853: 65-89Crossref PubMed Scopus (582) Google Scholar, 2Sandborg R.R. Smolen J.E. Lab. Invest. 1988; 59: 300-320PubMed Google Scholar, 3Seifert R. Schultz G. Rev. Physiol. Biochem. Pharmacol. 1991; 117: 1-338PubMed Google Scholar). The FPR belongs to the chemoattractant receptor family that is part of the superfamily of G-protein-coupled receptors (GPCRs) (4Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1122) Google Scholar, 5Kobilka B.K. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar). The FPR couples to the pertussis toxin (PTX)-sensitive G-proteins Gi2 and Gi3 (6Dillon S.B. Verghese M.W. Snyderman R. Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 1988; 55: 65-80PubMed Google Scholar, 7Klinker J.F. Wenzel-Seifert K. Seifert R. Gen. Pharmacol. 1996; 27: 33-54Crossref PubMed Scopus (91) Google Scholar). The stimulatory effects of FMLP are blocked by the competitive FPR antagonistsN-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine (BocPLPLP) and cyclosporin H (CsH) (8de Paulis A. Ciccarelli A. de Crescenzo G. Cirillo R. Patella V. Marone G. J. Allergy Clin. Immunol. 1996; 98: 152-164Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Kanaho Y. Kermode J.C. Becker E.L. J. Leukocyte Biol. 1990; 47: 420-428Crossref PubMed Scopus (5) Google Scholar, 10Wenzel-Seifert K. Grünbaum L. Seifert R. J. Immunol. 1991; 147: 1940-1946PubMed Google Scholar, 11Wenzel-Seifert K. Seifert R. J. Immunol. 1993; 150: 4591-4599PubMed Google Scholar). BocPLPLP is a synthetic linear peptide analogue of FMLP (9Kanaho Y. Kermode J.C. Becker E.L. J. Leukocyte Biol. 1990; 47: 420-428Crossref PubMed Scopus (5) Google Scholar), and CsH is a cyclic undecapeptide from the fungus Tolyplocadium inflatum Gams (12Traber R. Loosli H.-R. Hofmann H. Kuhn M. von Wartburg A. Helv. Chim. Acta. 1982; 65: 1655-1677Crossref Scopus (71) Google Scholar). Chemically, CsH is a stereoisomer of the immunosuppressant drug cyclosporin A (13von Wartburg A. Traber R. Prog. Allergy. 1986; 38: 28-45PubMed Google Scholar), but cyclosporin A is not an FPR antagonist (10Wenzel-Seifert K. Grünbaum L. Seifert R. J. Immunol. 1991; 147: 1940-1946PubMed Google Scholar,11Wenzel-Seifert K. Seifert R. J. Immunol. 1993; 150: 4591-4599PubMed Google Scholar).The two-state model of GPCR activation assumes that GPCRs exist in an inactive (“R”) state or an active (“R*”) state (14Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 15Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 16Schütz W. Freissmuth M. Trends Pharmacol. Sci. 1992; 13: 376-380Abstract Full Text PDF PubMed Scopus (145) Google Scholar). 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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 18Burstein E.S. Spalding T.A. Brann M.R. Mol. Pharmacol. 1997; 51: 312-319Crossref PubMed Scopus (111) Google Scholar).Many wild-type GPCRs including the β2-adrenoreceptor (19Bond 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. Lefkowitz R.J. Nature. 1995; 374: 272-276Crossref PubMed Scopus (409) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar), the 5-hydroxytryptamine2C receptor (22Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar), the histamine H2 receptor (23Smit M.J. Leurs R. Alewijnse A.E. Blauw J. Van Nieuw Amerongen G.P. Van De Vrede Y. Roovers E. Timmerman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6802-6807Crossref PubMed Scopus (210) Google Scholar), the δ-opioid receptor (24Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (477) Google Scholar), the thyrotropin receptor (25Cetani F. Tonacchera M. Vassart G. FEBS Lett. 1996; 378: 27-31Crossref PubMed Scopus (77) Google Scholar) and the α2D-adrenoreceptor (26Tian W.N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar) 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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 27Kenakin T. Trends Pharmacol. Sci. 1997; 18: 456-464Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 28Jakubik J. Bacakova L. el-Fakahany E.E. Tucek S. FEBS Lett. 1995; 377: 275-279Crossref PubMed Scopus (75) Google Scholar).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 (29Gierschik P. Sidiropoulos D. Steisslinger M. Jakobs K.H. Eur. J. Pharmacol. 1989; 172: 481-492Crossref PubMed Scopus (52) Google Scholar, 30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar, 31Klinker J.F. Schwaner I. Offermanns S. Hagelüken A. Seifert R. Biochem. Pharmacol. 1994; 48: 1857-1864Crossref PubMed Scopus (16) Google Scholar). Additionally, Na+ efficiently reduces basal G-protein activity in membranes expressing Gi-protein-linked constitutively active GPCRs (26Tian W.N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar, 32Costa T. Lang J. Gless C. Herz A. Mol. Pharmacol. 1990; 37: 383-394PubMed Google Scholar) and in myeloid membranes expressing FPR (29Gierschik P. Sidiropoulos D. Steisslinger M. Jakobs K.H. Eur. J. Pharmacol. 1989; 172: 481-492Crossref PubMed Scopus (52) Google Scholar, 30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar).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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 33Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 34Leopoldt D. Harteneck C. Nürnberg B. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 216-224Crossref PubMed Scopus (90) Google Scholar) and is highly sensitive at unmasking constitutive activity of GPCRs, provided that the appropriate complement of G-proteins is expressed (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar,35Barr A.J. Manning D.R. J. Biol. Chem. 1997; 272: 32979-32987Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 36Hartman J.I., IV Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 37Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Abstract Full Text PDF PubMed Google Scholar). Ye and colleagues (37Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Abstract Full Text PDF PubMed Google Scholar) 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, Giα2β1γ2, 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 (38Buhl A.M. Osawa S. Johnson G.L. J. Biol. Chem. 1995; 270: 19828-19832Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 39Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar). As read-out for FPR activity, we monitored guanosine 5′-O-(3-thiotriposphate) (GTPγS) binding to G-proteins (30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar, 35Barr A.J. Manning D.R. J. Biol. Chem. 1997; 272: 32979-32987Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 36Hartman J.I., IV Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Human neutrophils play a key role in host defense against bacterial infections (1Rossi F. Biochim. Biophys. Acta. 1986; 853: 65-89Crossref PubMed Scopus (582) Google Scholar, 2Sandborg R.R. Smolen J.E. Lab. Invest. 1988; 59: 300-320PubMed Google Scholar, 3Seifert R. Schultz G. Rev. Physiol. Biochem. Pharmacol. 1991; 117: 1-338PubMed Google Scholar). Neutrophils are activated by the bacterial formyl peptideN-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 (1Rossi F. Biochim. Biophys. Acta. 1986; 853: 65-89Crossref PubMed Scopus (582) Google Scholar, 2Sandborg R.R. Smolen J.E. Lab. Invest. 1988; 59: 300-320PubMed Google Scholar, 3Seifert R. Schultz G. Rev. Physiol. Biochem. Pharmacol. 1991; 117: 1-338PubMed Google Scholar). The FPR belongs to the chemoattractant receptor family that is part of the superfamily of G-protein-coupled receptors (GPCRs) (4Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1122) Google Scholar, 5Kobilka B.K. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar). The FPR couples to the pertussis toxin (PTX)-sensitive G-proteins Gi2 and Gi3 (6Dillon S.B. Verghese M.W. Snyderman R. Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 1988; 55: 65-80PubMed Google Scholar, 7Klinker J.F. Wenzel-Seifert K. Seifert R. Gen. Pharmacol. 1996; 27: 33-54Crossref PubMed Scopus (91) Google Scholar). The stimulatory effects of FMLP are blocked by the competitive FPR antagonistsN-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine (BocPLPLP) and cyclosporin H (CsH) (8de Paulis A. Ciccarelli A. de Crescenzo G. Cirillo R. Patella V. Marone G. J. Allergy Clin. Immunol. 1996; 98: 152-164Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Kanaho Y. Kermode J.C. Becker E.L. J. Leukocyte Biol. 1990; 47: 420-428Crossref PubMed Scopus (5) Google Scholar, 10Wenzel-Seifert K. Grünbaum L. Seifert R. J. Immunol. 1991; 147: 1940-1946PubMed Google Scholar, 11Wenzel-Seifert K. Seifert R. J. Immunol. 1993; 150: 4591-4599PubMed Google Scholar). BocPLPLP is a synthetic linear peptide analogue of FMLP (9Kanaho Y. Kermode J.C. Becker E.L. J. Leukocyte Biol. 1990; 47: 420-428Crossref PubMed Scopus (5) Google Scholar), and CsH is a cyclic undecapeptide from the fungus Tolyplocadium inflatum Gams (12Traber R. Loosli H.-R. Hofmann H. Kuhn M. von Wartburg A. Helv. Chim. Acta. 1982; 65: 1655-1677Crossref Scopus (71) Google Scholar). Chemically, CsH is a stereoisomer of the immunosuppressant drug cyclosporin A (13von Wartburg A. Traber R. Prog. Allergy. 1986; 38: 28-45PubMed Google Scholar), but cyclosporin A is not an FPR antagonist (10Wenzel-Seifert K. Grünbaum L. Seifert R. J. Immunol. 1991; 147: 1940-1946PubMed Google Scholar,11Wenzel-Seifert K. Seifert R. J. Immunol. 1993; 150: 4591-4599PubMed Google Scholar). The two-state model of GPCR activation assumes that GPCRs exist in an inactive (“R”) state or an active (“R*”) state (14Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 15Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 16Schütz W. Freissmuth M. Trends Pharmacol. Sci. 1992; 13: 376-380Abstract Full Text PDF PubMed Scopus (145) Google Scholar). 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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 18Burstein E.S. Spalding T.A. Brann M.R. Mol. Pharmacol. 1997; 51: 312-319Crossref PubMed Scopus (111) Google Scholar). Many wild-type GPCRs including the β2-adrenoreceptor (19Bond 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. Lefkowitz R.J. Nature. 1995; 374: 272-276Crossref PubMed Scopus (409) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar), the 5-hydroxytryptamine2C receptor (22Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar), the histamine H2 receptor (23Smit M.J. Leurs R. Alewijnse A.E. Blauw J. Van Nieuw Amerongen G.P. Van De Vrede Y. Roovers E. Timmerman H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6802-6807Crossref PubMed Scopus (210) Google Scholar), the δ-opioid receptor (24Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (477) Google Scholar), the thyrotropin receptor (25Cetani F. Tonacchera M. Vassart G. FEBS Lett. 1996; 378: 27-31Crossref PubMed Scopus (77) Google Scholar) and the α2D-adrenoreceptor (26Tian W.N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar) 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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 27Kenakin T. Trends Pharmacol. Sci. 1997; 18: 456-464Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 28Jakubik J. Bacakova L. el-Fakahany E.E. Tucek S. FEBS Lett. 1995; 377: 275-279Crossref PubMed Scopus (75) Google Scholar). 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 (29Gierschik P. Sidiropoulos D. Steisslinger M. Jakobs K.H. Eur. J. Pharmacol. 1989; 172: 481-492Crossref PubMed Scopus (52) Google Scholar, 30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar, 31Klinker J.F. Schwaner I. Offermanns S. Hagelüken A. Seifert R. Biochem. Pharmacol. 1994; 48: 1857-1864Crossref PubMed Scopus (16) Google Scholar). Additionally, Na+ efficiently reduces basal G-protein activity in membranes expressing Gi-protein-linked constitutively active GPCRs (26Tian W.N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar, 32Costa T. Lang J. Gless C. Herz A. Mol. Pharmacol. 1990; 37: 383-394PubMed Google Scholar) and in myeloid membranes expressing FPR (29Gierschik P. Sidiropoulos D. Steisslinger M. Jakobs K.H. Eur. J. Pharmacol. 1989; 172: 481-492Crossref PubMed Scopus (52) Google Scholar, 30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar). 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 (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 33Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 34Leopoldt D. Harteneck C. Nürnberg B. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 216-224Crossref PubMed Scopus (90) Google Scholar) and is highly sensitive at unmasking constitutive activity of GPCRs, provided that the appropriate complement of G-proteins is expressed (17Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 20Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 21Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar,35Barr A.J. Manning D.R. J. Biol. Chem. 1997; 272: 32979-32987Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 36Hartman J.I., IV Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 37Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Abstract Full Text PDF PubMed Google Scholar). Ye and colleagues (37Quehenberger O. Prossnitz E.R. Cochrane C.G. Ye R.D. J. Biol. Chem. 1992; 267: 19757-19760Abstract Full Text PDF PubMed Google Scholar) 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, Giα2β1γ2, 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 (38Buhl A.M. Osawa S. Johnson G.L. J. Biol. Chem. 1995; 270: 19828-19832Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 39Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar). As read-out for FPR activity, we monitored guanosine 5′-O-(3-thiotriposphate) (GTPγS) binding to G-proteins (30Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (83) Google Scholar, 35Barr A.J. Manning D.R. J. Biol. Chem. 1997; 272: 32979-32987Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 36Hartman J.I., IV Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 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 Giα2 baculovirus; to Dr. P. Gierschik for donating β1γ2 baculovirus; and Drs. B. Nürnberg and G. Schultz for providing anti-Giαcommon and anti-Gβcommon 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.