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• W2076460438 abstract "The human 5-hydroxytryptamine1Areceptor, when expressed in Spodoptera frugiperda (Sf9) cells, facilitates the binding of [35S]GTPγS to a co-expressed GTP-binding regulatory protein, Gz, consistent with constitutive activity. The antagonists 4-(2′-methoxyphenyl)-1-[2′(n-2“-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI) and the related fluorobenzamido analogue p-MPPF had little (p-MPPI) or no (p-MPPF) effect on this activity. In contrast, a third antagonist, the neuroleptic spiperone, produced an almost complete suppression. Thus, using G protein activation as an index of receptor activity, p-MPPF was classified as a neutral antagonist,p-MPPI as a partial inverse agonist, and spiperone as essentially a full inverse agonist. As predicted, spiperone displayed properties consistent with a special form of noncompetitive antagonism when used to displace the agonist [125I]R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin. Our data profile Sf9 cells as a unique vehicle for the characterization of inverse agonists, as these cells support a systematic pairing of mammalian receptors and G proteins, quantitative assays of G protein activation, and unambiguously labeled populations of coupled and uncoupled receptors. The human 5-hydroxytryptamine1Areceptor, when expressed in Spodoptera frugiperda (Sf9) cells, facilitates the binding of [35S]GTPγS to a co-expressed GTP-binding regulatory protein, Gz, consistent with constitutive activity. The antagonists 4-(2′-methoxyphenyl)-1-[2′(n-2“-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI) and the related fluorobenzamido analogue p-MPPF had little (p-MPPI) or no (p-MPPF) effect on this activity. In contrast, a third antagonist, the neuroleptic spiperone, produced an almost complete suppression. Thus, using G protein activation as an index of receptor activity, p-MPPF was classified as a neutral antagonist,p-MPPI as a partial inverse agonist, and spiperone as essentially a full inverse agonist. As predicted, spiperone displayed properties consistent with a special form of noncompetitive antagonism when used to displace the agonist [125I]R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin. Our data profile Sf9 cells as a unique vehicle for the characterization of inverse agonists, as these cells support a systematic pairing of mammalian receptors and G proteins, quantitative assays of G protein activation, and unambiguously labeled populations of coupled and uncoupled receptors. The 5-hydroxytryptamine1A(5-HT1A) 1The abbreviations used are: 5-HT1A, 1A subtype of 5-hydroxytryptamine; 5-HT, 5-hydroxytryptamine (serotonin); 8-OH-PIPAT,R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin; G protein, GTP-binding regulatory protein; GPCR, G protein-coupled receptor; GTPγS, guanosine 5′-3-O-(thio)triphosphate;p-MPPF, 4-(2′-methoxyphenyl)-1-[2′-(n-2“-pyridinyl)-p-fluorobenzamido]ethyl-piperazine;p-MPPI, 4-(2′-methoxyphenyl)-1-[2′-(n-2”-pyridinyl)-p-iodobenzamido]ethyl-piperazine; Sf9, Spodoptera frugiperda. receptor is one of several subtypes of receptors for serotonin (5-HT) that mediate the actions of this agonist on neuronal activity. The 5-HT1A receptor exists at particularly high densities in the hippocampus and components of the limbic system and can be divided functionally into two populations – somatodendritic autoreceptors in the dorsal raphe nucleus, which mediate the stimulation of K+ channels, and postsynaptic receptors elsewhere, which mediate both the stimulation of K+ channels and inhibition of adenylyl cyclase (reviewed by Hoyer et al. (1Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar)). Other activities ascribed to the 5-HT1A receptor include the activation of the mitogen-activated protein kinases ERK1 and ERK2 (2Cowen D.S. Sowers R.S. Manning D.R. J. Biol. Chem. 1996; 271: 22297-22300Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), activation of nuclear factor-κB (3Cowen D.S. Molinoff P.B. Manning D.R. Mol. Pharmacol. 1997; 52: 221-226Crossref PubMed Scopus (47) Google Scholar), and stimulation of Na+/H+ exchange (4Garnovskaya M.N. Gettys T.W. van Biesen T. Prpic V. Chuprun J.K. Raymond J.R. J. Biol. Chem. 1997; 272: 7770-7776Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). All of these actions appear to be exerted through one or more members of the Gifamily of GTP-binding regulatory proteins (G proteins), which comprises Gi, Go, and Gz (5Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (204) Google Scholar). The 5-HT1A receptor can also interact to some extent with members of the Gq family to achieve activation of a phosphoinositide-specific phospholipase C depending on the cell and/or concentration of agonist (6Fargin A. Raymond J.R. Regan J.W. Cotecchia S. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1989; 264: 14848-14852Abstract Full Text PDF PubMed Google Scholar, 7Liu Y.F. Albert P.R. J. Biol. Chem. 1991; 266: 23689-23697Abstract Full Text PDF PubMed Google Scholar). The deduced structure of the 5-HT1A receptor resembles that of the many other G protein-coupled receptors (GPCRs) now identified (8Kobilka B.K. Frielle T. Collins S. Yang-Feng T. Kobilka T.S. Francke U. Lefkowitz R.J. Caron M.G. Nature. 1987; 329: 75-79Crossref PubMed Scopus (432) Google Scholar, 9Fargin A. Raymond J.R. Lohse M.J. Kobilka B.K. Caron M.G. Lefkowitz R.J. Nature. 1988; 335: 358-360Crossref PubMed Scopus (525) Google Scholar). The 5-HT1A receptor is a prime target in the development of therapeutic agents for the treatment of affective disorders such as anxiety and depression (10Fletcher A. Cliffe I.A. Dourish C.T. Trends Pharmacol. Sci. 1993; 14: 441-448Abstract Full Text PDF Scopus (128) Google Scholar). Not suprisingly, a vast array of ligands—agonists, partial agonists, and antagonists—have been generated (1Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar, 10Fletcher A. Cliffe I.A. Dourish C.T. Trends Pharmacol. Sci. 1993; 14: 441-448Abstract Full Text PDF Scopus (128) Google Scholar). Among agonists with a particularly high degree of selectivity are 8-hydroxy-N,N-dipropyl-2-aminotetralin and R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin (8-OH-PIPAT) (11Gozlan H. Mestikawy S.E. Pichat L. Glowinski J. Hamon M. Nature. 1983; 305: 140-142Crossref PubMed Scopus (717) Google Scholar, 12Zhuang Z.-P. Kung M.-P. Kung H.F. J. Med. Chem. 1993; 36: 3161-3165Crossref PubMed Scopus (36) Google Scholar). Antagonists of a similar selectivity include 4-(2′-methoxyphenyl)-1-[2′-(n-2“-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI) and the related fluorobenzamido analogue (p-MPPF) (13Zhuang Z.-P. Kung M.-P. Kung H.F. J. Med. Chem. 1994; 37: 1406-1407Crossref PubMed Scopus (109) Google Scholar, 14Kung H.F. Kung M.-P. Clarke W. Maayani S. Zhuang Z.-P. Life Sci. 1994; 55: 1459-1462Crossref PubMed Scopus (61) Google Scholar, 15Kung M.-P. Frederick D. Mu M. Zhuang Z.-P. Kung H.F. J. Pharmacol. Exp. Ther. 1995; 272: 429-437PubMed Google Scholar, 16Kung H.F. Stevenson D.A. Zhuang Z.-P. Kung M.-P. Frederick D. Hurt S.D. Synapse. 1996; 23: 344-346Crossref PubMed Scopus (50) Google Scholar). The neuroleptic spiperone is also a commonly employed antagonist for the 5-HT1A receptor, although it blocks a number of other receptors as well (17Leysen J.E. Gommeren W. Laduron P.M. Biochem. Pharmacol. 1978; 27: 307-316Crossref PubMed Scopus (435) Google Scholar, 18Leysen J.E. Niemegeers C.J.E. Tollenaere J.P. Laduron P.M. Nature. 1978; 272: 168-171Crossref PubMed Scopus (547) Google Scholar). Curiously, spiperone exhibits a wide variation in K i values depending on the mode of assay, and in particular on whether it competes with radiolabeled agonists, in the presence or absence of GTP or radiolabeled antagonists (9Fargin A. Raymond J.R. Lohse M.J. Kobilka B.K. Caron M.G. Lefkowitz R.J. Nature. 1988; 335: 358-360Crossref PubMed Scopus (525) Google Scholar, 15Kung M.-P. Frederick D. Mu M. Zhuang Z.-P. Kung H.F. J. Pharmacol. Exp. Ther. 1995; 272: 429-437PubMed Google Scholar, 19Hall M.D. El Mestikawy S. Emerit M.B. Pichat L. Hamon M. Gozlan H. J. Neurochem. 1985; 44: 1685-1696Crossref PubMed Scopus (441) Google Scholar, 20Peroutka S.J. J. Neurochem. 1986; 47: 529-540Crossref PubMed Scopus (354) Google Scholar, 21Hensler J.G. Cervera L.S. Miller H.A. Corbitt J. J. Pharmacol. Exp. Ther. 1996; 278: 1138-1145PubMed Google Scholar, 22Albert P.R. Zhou Q.-Y. Van Tol H.H.M. Bunzow J.R. Civelli O. J. Biol. Chem. 1990; 265: 5825-5832Abstract Full Text PDF PubMed Google Scholar, 23Guan X.-M. Peroutka S.J. Kobilka B.K. Mol. Pharmacol. 1992; 41: 695-698PubMed Google Scholar, 24Sundaram H. Newman-Tancredi A. Strange P.G. Biochem. Pharmacol. 1993; 45: 1003-1009Crossref PubMed Scopus (48) Google Scholar, 25Raymond J.R. Fargin A. Lohse M.J. Regan J.W. Senogles S.E. Lefkowitz R.J. Caron M.G. Mol. Pharmacol. 1989; 36: 15-21PubMed Google Scholar). The classical model of GPCR action implies that the binding of an agonist to a receptor, e.g. serotonin to the 5-HT1A receptor, is essential for activation of the receptor and transmission of the biological signal across the plasma membrane. However, studies of several GPCRs operating through distinct G proteins have revealed that receptor activation can occur to some extent spontaneously in the absence of an agonist. Agonist-independent, or constitutive, activity has been reported for the δ-opioid, α2-adrenergic, and muscarinic cholinergic receptors expressed normally (26Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (479) Google Scholar, 27Tian W.N. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar, 28Hilf G. Jakobs K.H. Eur. J. Pharmacol. 1992; 225: 245-252Crossref PubMed Scopus (73) Google Scholar) and for the dopamine D5 (29Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar), β2-adrenergic (30Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar), 5-HT1B (31Thomas D.R. Faruq S.A. Balcarek J.M. Brown A.M. J. Recept. Sign. Transd. Res. 1995; 15: 199-211Crossref PubMed Scopus (73) Google Scholar), 5-HT1D (31Thomas D.R. Faruq S.A. Balcarek J.M. Brown A.M. J. Recept. Sign. Transd. Res. 1995; 15: 199-211Crossref PubMed Scopus (73) Google Scholar), and 5-HT2C (32Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar) receptors overexpressed in mammalian cells following transfection. Overexpression of the β2-adrenergic receptor in a transgenic mouse model also elicits a marked set of agonist-independent responses (33Milano C.A. Allen L.F. Rockman H.A. Dolber P.C. McMinn T.R. Chien K.R. Johnson T.D. Bond R.A. Lefkowitz R.J. Science. 1994; 264: 582-586Crossref PubMed Scopus (662) Google Scholar). Mutations in GPCRs, introduced near the junction of the third cytoplasmic loop and transmembrane domain 6 (30Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 34Kjelsberg M.A. Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1430-1433Abstract Full Text PDF PubMed Google Scholar) or occurring naturally throughout the receptor (35Robinson P.R. Cohen G.B. Zhukovsky E.A. Oprian D.D. Neuron. 1992; 9: 719-725Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 36Robbins L.S. Nadeau J.H. Johnson K.R. Kelly M.A. Roselli-Rehfuss L. Baack E. Mountjoy K.G. Cone R.D. Cell. 1993; 72: 827-834Abstract Full Text PDF PubMed Scopus (775) Google Scholar), can dramatically enhance constitutive activity. In part to account for constitutive activity but also to explain changes in ligand affinity as a function of conformations underlying this activity, the ternary complex model has been revised to include an equilibrium between inactive (R) and active (R*) conformations of the receptor (37Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 38Kenakin T. Pharmacol. Rev. 1996; 48: 413-463PubMed Google Scholar). The active conformation, which through the established equilibrium can exist in some small amount without agonist, is capable of binding to and activating a relevant G protein. Agonists bind preferentially to R* and/or R*G, and thereby shift the equilibrium toward R* as represented by agonist·R* and agonist·R*·G complexes. The discovery of constitutive receptor activity has opened the way for reclassification of “antagonists” as neutral antagonists and inverse agonists (sometimes called reverse antagonists). Neutral antagonists, through competition with agonists for binding to the receptor, block the actions of agonists but have no effect on constitutive activity. Neutral antagonists are thought to bind R and R* in an equivalent fashion, thereby preserving the existing equilibrium between these two forms of receptor. Inverse agonists not only block the actions of agonists but suppress constitutive activity. Just as agonists shift the equilibrium toward R* and R*G by selectively binding R* forms of the receptor, inverse agonists may shift the equilibrium toward R by binding the R form of receptor in preference to R* or R*G. Inverse agonism was first recognized as a property of β-carbolines at the unrelated GABAA (γ-aminobutyric acid) receptor (39Braestrup C. Schmiechen R. Neef G. Nielsen M. Petersen E.N. Science. 1982; 216: 1241-1243Crossref PubMed Scopus (498) Google Scholar) and has subsequently been identified for a number of GPCRs (26Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (479) Google Scholar, 32Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar,40Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 41Labrecque J. Fargin A. Bouvier M. Chidiac P. Dennis M. Mol. Pharmacol. 1995; 48: 150-159PubMed Google Scholar, 42Samama P. Pei G. Costa T. Cotecchia S. Lefkowitz R.J. Mol. Pharmacol. 1994; 45: 390-394PubMed Google Scholar, 43Bond 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 (410) Google Scholar). Spodoptera frugiperda (Sf9) cells represent a powerful model with which to characterize the activity of mammalian GPCRs (44Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: 519-527Abstract Full Text PDF PubMed Google Scholar, 45Mulheron J.G. Casanas S.J. Arthur J.M. Garnovskaya M.N. Gettys T.W. Raymond J.R. J. Biol. Chem. 1994; 269: 12954-12962Abstract Full Text PDF PubMed Google Scholar, 46Parker E.M. Grisel D.A. Iben L.G. Nowak H.P. Mahle C.D. Yocca F.D. Gaughan G.T. Eur. J. Pharmacol. 1994; 268: 43-53Crossref PubMed Scopus (30) Google Scholar, 47Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 48Hartman J.L. Northup J.K. J. Biol. Chem. 1996; 271: 22591-22597Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Receptors, together with selected G protein α, β, and γ subunits, can be co-expressed in intact Sf9 cells through infection with appropriate recombinant baculoviruses. Receptor activity (with or without agonist) can then be characterized in subsequently isolated membranes as a facilitation of [35S]GTPγS binding to the G protein α subunit (49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The protocol of reconstitution and assay using Sf9 cells is particularly advantageous for the reasons that the pairing of receptor with G protein can be defined precisely, that little or no interference is imposed by endogenous receptors and G proteins, and that G protein activation is a more direct correlate of intrinsic efficacy than effector activity. We demonstrated previously the expected selectivity in receptor·G protein coupling for several receptors working through one or more of the four families of G proteins (49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Of interest in the previous study was the observation that the 5-HT1A receptor alone of the receptors examined expressed a perceptible level of what appeared to be constitutive activity. We confirm here that the 5-HT1A receptor exhibits constitutive activity and, using this activity as a starting point, demonstrate that the Sf9 cell reconstitution model can provide a direct and quantifiable assay to distinguish neutral antagonists from inverse agonists. We find that p-MPPF is a neutral antagonist, whereas p-MPPI and spiperone, respectively, are partial and full inverse agonists. We also describe the means by which unambiguously defined populations of uncoupled and coupled receptor can be constructed in Sf9 cells, and we provide definitions of efficacy based on G protein activation and ligand displacement analysis. Recombinant baculoviruses encoding the G protein subunits β1 and γ2 (50Iniguez-Lluhi J.A. Simon M.I. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1992; 267: 23409-23417Abstract Full Text PDF PubMed Google Scholar) were kindly provided by Drs. T. Kozasa and A. Gilman at Southwestern Medical Center (Dallas, TX). Those encoding the 5-HT1A receptor and αz were constructed in our laboratory (47Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The procedures for handling Sf9 cells and assaying [35S]GTPγS binding have been described previously (47Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Essentially, Sf9 cells were propagated in suspension culture with TNM-FH medium containing charcoal-treated serum. For infection with recombinant baculoviruses, the cells were subcultured in monolayer and infected with one or more viruses at a multiplicity of infection of at least one for each virus. The medium was replaced 16 h following infection with Sf900II optimized serum-free medium (Life Technologies, Inc.). The cells were harvested at 48 h, and membranes were prepared by differential pelleting following lysis of the cells under hypotonic conditions. [35S]GTPγS binding was assayed by incubation of the membranes (20 μg of protein per assay point) with or without agonists and/or antagonists for 5 min at 30 °C with 30 nm[35S]GTPγS (1300 Ci/mmol) in the presence of 1 μm GDP and 3 mm free Mg2+. In experiments with antagonists, membranes were preincubated for 10 min at room temperature with the antagonists prior to initiating the binding assay. Immunoprecipitation of αz was accomplished with antiserum 6354 (directed toward residues 24–33 (51Lounsbury K.M. Casey P.J. Brass L.F. Manning D.R. J. Biol. Chem. 1991; 266: 22051-22056Abstract Full Text PDF PubMed Google Scholar)) following extraction of the membranes with 0.5% Nonidet P-40. Bound radioactivity was quantitated by scintillation spectrometry. The procedures of analysis were those of Kreiss et al. (52Kreiss D.S. Wieland S. Lucki I. Neuroscience. 1993; 52: 295-301Crossref PubMed Scopus (82) Google Scholar). Sf9 cell membranes were resuspended in 0.1% ice-cold perchloric acid and sonicated briefly. Membrane protein was pelleted by centrifugation at 16,000 × g for 5 min, and the supernatant was filtered through a 0.2 μm filter. Samples were applied to a Bioanalytical Systems 480 reverse-phase microbore column (1 × 100 mm), and fractions were analyzed with an LC-4C electrochemical detector. The mobile phase consisted of 0.67 mm EDTA, 0.43 mm sodium octyl sulfate, 10 mm NaCl, 32 mm NaH2PO4, and 11% acetonitrile, pH 4.0. 5-HT, which had a retention time of 8 min, was used as a standard. The limit of detection was 0.1 nm. Binding assays were carried out in glass tubes (12 × 75 mm) in a final volume of 100 μl of binding buffer containing 50 mm Tris-HCl, pH 7.4, 2.5 mmMgCl2, and 0.1% bovine serum albumin. In competition experiments, membranes (1–2 μg of protein per assay tube) were incubated for 40 min at 37 °C with 0.4 nm[125I]p-MPPI (2200 Ci/mmol) or 0.4 nm [125I]8-OH-PIPAT (2200 Ci/mmol) and the competing ligands. Assays were terminated by the addition of 5 ml of ice-cold wash buffer (50 mm Tris-HCl, pH 7.4). The reaction mixture was filtered through glass fiber filters (Schleicher and Schuell No. 32, previously soaked in 0.3% polyethelenimine), and the filters were washed with 15 ml of ice-cold wash buffer using a Brandel cell harvester. Filters were counted in a 1219 Wallac (Gaithersburg, MD) γ counter at an efficiency of 75%. Nonspecific binding for [125I]p-MPPI was defined with 30 μm spiperone or 100 μm 5-HT, and that for [125I]8-OH-PIPAT was defined with 10 μm5-HT. Specific binding of these compounds at their K d values was 70 and 78%, respectively. Curve fitting and linear regression were carried out using GraphPad Prism (ISI Software). One-site and two-site fits were compared by calculating the F ratio, with a p value of less than 0.05 considered to be significant. Parameter values are quoted as means ± S.E. 5-HT, p-MPPI, and spiperone were obtained from Research Biochemicals Inc. p-MPPF was a gift from Drs. H. F. Kung and M.-P. Kung at the University of Pennsylvania. The radiolabeled compounds were obtained from NEN Life Science Products or provided by Drs. H. F. Kung and M.-P. Kung. Sf9 cells expressing the human 5-HT1A receptor and Gz (i.e. αz, β1, and γ2) were used to explore the properties of compounds that bind to the 5-HT1A receptor. Gz is a member of the Gi family and has been used extensively by us to study the coupling of the receptor to G proteins (47Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The chief advantage of Gz is that it does not bind significant levels of [35S]GTPγS when expressed alone and thus affords a clear picture of activation achieved through the receptor. Activation is equated here with the binding of [35S]GTPγS to αz in Sf9 cell membranes as quantitated following immunoprecipitation. A fraction of Gz co-expressed with the 5-HT1Areceptor was found to assume an activated state in the presence of [35S]GTPγS but absence of added agonist (Fig.1, hatched column). As implied above, the activation was not achieved if Gz was expressed without the receptor (49Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Moreover, the activation was not the consequence of 5-HT carried over from the medium used in cell culture. Serum-free conditions were employed during the time at which the 5-HT1A receptor was expressed, and no 5-HT was detected in membranes by direct analysis. These data demonstrate that the 5-HT1A receptor expressed in Sf9 cells exhibits a discernible level of constitutive activity. The receptor is not fully active, however, as 5-HT promoted further activation of Gz(Fig. 1, curve). The activation by 5-HT was dose-dependent, characterized by an EC50(14 ± 6 nm) comparable to that determined in mammalian cells. The compounds p-MPPI, p-MPPF, and spiperone are known to be antagonists of 5-HT at the 5-HT1A receptor (13Zhuang Z.-P. Kung M.-P. Kung H.F. J. Med. Chem. 1994; 37: 1406-1407Crossref PubMed Scopus (109) Google Scholar, 14Kung H.F. Kung M.-P. Clarke W. Maayani S. Zhuang Z.-P. Life Sci. 1994; 55: 1459-1462Crossref PubMed Scopus (61) Google Scholar, 15Kung M.-P. Frederick D. Mu M. Zhuang Z.-P. Kung H.F. J. Pharmacol. Exp. Ther. 1995; 272: 429-437PubMed Google Scholar, 16Kung H.F. Stevenson D.A. Zhuang Z.-P. Kung M.-P. Frederick D. Hurt S.D. Synapse. 1996; 23: 344-346Crossref PubMed Scopus (50) Google Scholar). As shown in Fig. 2 A, all three compounds completely inhibited the activation of Gz evoked by 5-HT. IC50 values of p-MPPI, p-MPPF, and spiperone (at 100 nm 5-HT) were about 30, 40, and 310 nm, respectively. Of interest was the observation in these experiments that spiperone, and possibly to some extent p-MPPI, might actually suppress the activation of Gz to a level below that attributed to constitutive receptor activity. Such an action would represent a property of inverse agonism. The possibility was explored further, as shown for the receptor incubated without 5-HT in Fig.2 B. p-MPPF had no effect on constitutive activity at a concentration shown above to be sufficient to block the response to 100 nm 5-HT, and p-MPPI suppressed activity by only a small extent (16 ± 5%, which is significant (p < 0.01)). An almost complete suppression of constitutive activity, however, was achieved with spiperone. The IC50 for this action was 68 nm. These data imply that p-MPPF is a neutral antagonist, p-MPPI is a weak inverse agonist, and spiperone is a full inverse agonist. The effect of spiperone on constitutive receptor activity was completely reversed by p-MPPF (Fig.3). A similar reversal was achieved with p-MPPI, with the response returning to that observed in the presence of p-MPPI alone. These results confirm that the 5-HT1A receptor is the site of action for spiperone and that the constitutive activity of the receptor is not attributable to 5-HT carried over from the cell culture medium. Importantly, the data as a whole demonstrate that the direct activation of a G protein as reconstituted in Sf9 cells can serve as a monitor of constitutive activity and can thereby be used to distinguish inverse agonists from neutral antagonists. The ternary complex model predicts that spiperone, as an inverse agonist, would have a higher affinity for the receptor in the absence of Gz than in its presence, whereas a neutral antagonist would have an equal affinity in both cases. Initial binding experiments with [3H]spiperone were unsuccessful due to the high level of nonspecific binding at the high concentrations required. We therefore carried out competition assays using [125I]p-MPPI as the radioligand, on the basis that this compound is a nearly neutral antagonist and might bind to coupled and uncoupled forms of the receptor equivalently (the ready availability of p-MPPI over p-MPPF as a radioiodinated ligand was conducive to this and subsequent assays). Scatchard analysis of [125I]p-MPPI binding to Sf9 cell membranes expressing the 5-HT1A receptor alone and together with Gz gave K d values of 0.46 ± 0.06 and 0.51 ± 0.03 nm, respectively (n = 6) (data not shown). The binding of [125I]p-MPPI under the two conditions with apparently equal affinity provided the basis for determining whether other ligands might discriminate between coupled and uncoupled forms of receptor. Displacement of [125I]p-MPPI by 5-HT in the presence of Gz was best fit by a two-site model, wherein about 30% of the receptor exhibited high affinity for 5-HT (K i = 6 ± 3 nm), and the remainder exhibited low affinity (K i = 590 ± 60 nm) (Fig. 4 A). Despite the high level of expression of receptor and G protein, therefore, only one-third of the receptor was found to preexist in a coupled state or to enter into a coupled state consequent to binding 5-HT. In the absence of Gz, 5-HT displaced [125I]p-MPPI binding in a monophasic manner with uniformly low affinity (K i = 640 ± 100 nm). These data represent the first measurement of the “K H /K L ” ratio, a commonly employed index of efficacy, for 5-HT using a single G protein (the ratio is about 100). Displacement of [125I]p-MPPI by p-MPPF, as anticipated, was about the same regardless of G protein (K i = 3.5 ± 0.4 and 3.1 ± 0.3 nm in the absence and presence of G protein, respectively) (Fig. 4 B). Similar results were obtained using p-MPPI to displace [125I]p-MPPI (K i = 2.4 ± 0.3 and 2.4 ± 0.1 nm, respectively) (data not shown). To our initial surprise, displacement curves with spiperone were essentially unaffected by G protein (K i = 60 ± 6 nm and 83 ± 9 nm, respectively) (Fig.4 C). Assuming that Gz couples to only 30% of the receptor in the absence of agonist (it may well be less), a difference in affinity of spiperone for uncoupled and coupled forms of the receptor greater than ∼20–30-fold would be required for a biphasic displacement to be clearly resolved. An assay to selectively label the G protein-coupled form of receptor was therefore designed. For labeling, we used the agonist [125I]8-OH-PIPAT. [125I]8-OH-PIPAT binds the 5-HT1A receptor only in the presence of G protein (47Butkerait P. Zheng Y. Hallak H. Graham T.E. Miller H.A. Burris K.D. Molinoff P.B. Manning D.R. J. Biol. Chem. 1995; 270: 18691-18699Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). It binds the" @default.
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• W2076460438 title "Agonist-independent Activation of Gz by the 5-Hydroxytryptamine1A Receptor Co-expressed in Spodoptera frugiperda Cells" @default.
• W2076460438 cites W1489775551 @default.
• W2076460438 cites W1491140651 @default.
• W2076460438 cites W1492552461 @default.
• W2076460438 cites W1495834546 @default.
• W2076460438 cites W1500720615 @default.
• W2076460438 cites W1506946474 @default.
• W2076460438 cites W1510104405 @default.
• W2076460438 cites W1524415596 @default.
• W2076460438 cites W1536524213 @default.
• W2076460438 cites W1541533178 @default.
• W2076460438 cites W1604834232 @default.
• W2076460438 cites W1677997440 @default.
• W2076460438 cites W1964191104 @default.
• W2076460438 cites W1979113361 @default.
• W2076460438 cites W1979816107 @default.
• W2076460438 cites W1981047025 @default.
• W2076460438 cites W1981818755 @default.
• W2076460438 cites W1982527304 @default.
• W2076460438 cites W1982977937 @default.
• W2076460438 cites W1983515226 @default.
• W2076460438 cites W1983649348 @default.
• W2076460438 cites W1987339017 @default.
• W2076460438 cites W1990587716 @default.
• W2076460438 cites W1998317438 @default.
• W2076460438 cites W2003492314 @default.
• W2076460438 cites W2007537092 @default.
• W2076460438 cites W2012638283 @default.
• W2076460438 cites W2013968961 @default.
• W2076460438 cites W2017659544 @default.
• W2076460438 cites W2020697786 @default.
• W2076460438 cites W2021495235 @default.
• W2076460438 cites W2025237223 @default.
• W2076460438 cites W2031335522 @default.
• W2076460438 cites W2041987085 @default.
• W2076460438 cites W2043750066 @default.
• W2076460438 cites W2046209075 @default.
• W2076460438 cites W2062778364 @default.
• W2076460438 cites W2064483656 @default.
• W2076460438 cites W2077425788 @default.
• W2076460438 cites W2082016554 @default.
• W2076460438 cites W2088157575 @default.
• W2076460438 cites W2100059138 @default.
• W2076460438 cites W2119335473 @default.
• W2076460438 cites W2121031455 @default.
• W2076460438 cites W2158907779 @default.
• W2076460438 cites W4298998623 @default.
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