Evidence for Distinct Antagonist-Revealed Functional States of 5Hydroxytryptamine2A Receptor Homodimers

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Evidence for Distinct Antagonist-Revealed Functional States of 5Hydroxytryptamine2A Receptor Homodimers
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  MOL #54395 1 Evidence for distinct antagonist-revealed functional states of 5-HT 2A  receptor homodimers José Brea 1 , Marián Castro 1 , Jesús Giraldo 1  , Juan F. López-Giménez, Juan Fernando Padín, Fátima Quintián, Maria Isabel Cadavid, Maria Teresa Vilaró, Guadalupe Mengod, Kelly A. Berg, William P. Clarke, Jean-Pierre Vilardaga, Graeme Milligan and Maria Isabel Loza   Departamento de Farmacología, Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain (J. B., M.C., J. F. P., F. Q., M. I. C. and M. I. L.); Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain (J. G.); Department of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, UK (J. F. L-G. and G. Milligan); Department of Neurochemistry and Neuropharmacology. Instituto de Investigaciones Biomédicas de Barcelona, CSIC, IDIBAPS, Barcelona, Spain (M. T. V. and G. Mengod); Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229, USA (W. P. C. and K. A. B.); Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center, Pittsburgh, PA, 15213 and; Endocrine Unit, Massachussets General Hospital and Harvard Medical School, Boston MA 02114, USA (J. –P.V.) Molecular Pharmacology Fast Forward. Published on March 11, 2009 as doi:10.1124/mol.108.054395   Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics. This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on March 11, 2009 as DOI: 10.1124/mol.108.054395   a  t  A S P E T  J   o ur n a l   s  on S  e  p t   e m b  e r 1  9  ,2  0 1  6 m ol   ph  a r m . a  s  p e  t   j   o ur n a l   s  . or  gD o wnl   o a  d  e  d f  r  om   MOL #54395 2  Running title : Effector-related differences in 5-HT 2A  receptor homodimers. Corresponding author:  María Isabel Loza, Dpto. Farmacología. Facultad de Farmacia. Campus Sur. 15782 Santiago de Compostela, Spain., Phone: 34-981 547 139, Fax: 34-981 594 595, email: mabel.loza@usc.es. Number of: -text pages : 27 - tables : 1 - figures : 6 - references : 61 Number of words in : - Abstract : 104 - Introduction : 681 - Discussion : 1584 Abbreviations : 5-HT, serotonin; AA, arachidonic acid; BSA, bovine serum albumin; CHO, chinese hamster ovary; DMEM, Dulbecco’s modified Eagle’s medium; DOB, ((±))-1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane; DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; FCS, fetal calf serum; GR55562, (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenylbenzamide dihydrobromide; GPCR, G protein-coupled receptor; HBSS, Hank’s balanced salt solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid; IP, inositol phosphates; MDL100,907, (  R )-(±)-4-[1-hydroxy-1(2,3-dimethoxyphenyl)metyhyl]  N  -2-4-fluorophenylethyl)piperidine; PBS, phosphate buffered saline; PLA 2 , phospholipase A 2 ; PLC, phospholipase C. This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on March 11, 2009 as DOI: 10.1124/mol.108.054395   a  t  A S P E T  J   o ur n a l   s  on S  e  p t   e m b  e r 1  9  ,2  0 1  6 m ol   ph  a r m . a  s  p e  t   j   o ur n a l   s  . or  gD o wnl   o a  d  e  d f  r  om   MOL #54395 3 Abstract The serotonin (5-hydroxytryptamine, or 5-HT) 2A receptor is a cell surface class A G protein-coupled receptor that regulates a multitude of physiological functions of the body, and is a target for antipsychotic drugs. Here we found by means of FRET and immunoprecipitation studies that the 5-HT 2A -receptor homo-dimerized in live cells, which we linked with its antagonist-dependent fingerprint in both binding and receptor signaling. Some antagonists, like the atypical antipsychotics clozapine and risperidone, differentiate themselves from others, like the typical antipsychotic haloperidol, antagonizing these 5-HT 2A  receptor-mediated functions in a pathway-specific manner, explained here by a new model of multiple active interconvertible conformations at dimeric receptors. This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on March 11, 2009 as DOI: 10.1124/mol.108.054395   a  t  A S P E T  J   o ur n a l   s  on S  e  p t   e m b  e r 1  9  ,2  0 1  6 m ol   ph  a r m . a  s  p e  t   j   o ur n a l   s  . or  gD o wnl   o a  d  e  d f  r  om   MOL #54395 4 G protein-coupled receptors (GPCRs) constitute the major family of cell surface proteins involved in cell signaling cascades, and are the target of ≈  50% of clinical drugs (Imming et al., 2006). Studies on ligand-GPCR interactions performed over the last decade have revealed diverse capacities of ligand-GPCR-effector complexes to fine-tune their own signals, broadening its apparent simplicity and highlighting ligands as individual chemical species capable of transmitting messages into cellular function with a versatility unpredicted two decades ago (Kenakin, 2007b;Urban et al., 2007). It is well accepted that agonist (full and partial) ligands and allosteric positive regulators can invoke different active conformations of GPCRs and that these may allow differential agonist-dependent regulation of signaling pathways. Such effects have been described as ‘agonist-directed trafficking of receptor stimulus’ (Kenakin, 1995), ‘biased agonism’ (Jarpe et al., 1998), ‘functional selectivity’ (Urban et al., 2007) or ‘collateral efficacy’ (Kenakin, 2007a). This recently accumulated experimental evidence has led to the development of novel mathematical representations that attempt to explain the chemical biology of GPCRs and integrate the new knowledge by extending accepted traditional models (De Lean A. et al., 1980;Kent et al., 1980;Leff, 1995;Leff et al., 1997;Lefkowitz et al., 1993;Samama et al., 1993;Scaramellini and Leff, 2002;Weiss et al., 1996). Although it is generally believed that antagonists (neutral antagonists and inverse agonists) simply inhibit either agonist-induced or constitutive receptor functions, it is conceptually plausible to envision that certain antagonists could also deactivate GPCR responses in a ligand- and pathway-specific manner. A series of observations including the ability of certain ligands that are conventionally described as ‘antagonists’ to induce receptor internalization (Baker and Hill, 2007;Kenakin, 2007b), to cause activation of ERK MAP kinase (Wisler et al., 2007;Azzi et al., 2003) or to promote inverse agonist- This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on March 11, 2009 as DOI: 10.1124/mol.108.054395   a  t  A S P E T  J   o ur n a l   s  on S  e  p t   e m b  e r 1  9  ,2  0 1  6 m ol   ph  a r m . a  s  p e  t   j   o ur n a l   s  . or  gD o wnl   o a  d  e  d f  r  om   MOL #54395 5 specific receptor conformational changes (Vilardaga et al., 2005) are consistent with such a concept. This is particularly relevant because most current drugs that target GPCRs are antagonists. In the last decade there has been growing evidence to indicate that GPCR dimerization may be a requisite for function and that binding of small ligands to these receptors occurs on dimeric receptor forms (Ayoub et al., 2002;Herrick-Davis et al., 2005;Milligan, 2004), although monomeric forms of GPCRs have also been shown to be capable of activating G proteins as well (Bayburt et al., 2007;Whorton et al., 2007), suggesting that functionally active monomeric and dimeric forms of the receptors may coexist in equilibrium. Mechanisms by which ligands differentially regulate signaling pathways mediated by a single receptor generally considered the ability of ligands to differentially stabilize distinct receptor conformations (Hunton et al., 2005). The question is to what extent these different conformations occur at a single or at a paired receptor .  The 5-HT 2A  receptor is a class A GPCR whose antagonists have important applications in the treatment of disorders of the cardiovascular and central nervous systems (Berg et al., 2005), and also in virology (Elphick et al., 2004). 5-HT 2A  receptors regulate IP accumulation mediated by phospholipase C and AA release mediated at least partially by phospholipase A 2 , and different 5-HT 2A  receptor agonists, for instance serotonin and the hallucinogenic (±)DOI (2,5-dimethoxy-4-iodoamphetamine), show functional selectivity discriminating between these two signaling pathways (Berg et al., 1998).   In a previous study (Lopez-Gimenez et al., 2001), in native human brain and in cell lines expressing recombinant human 5-HT 2A  receptors lacking constitutive activity, we observed Gpp(NH)p-independent shallow, biphasic, competition binding curves for antagonists competing with agonist radioligands. In light of current knowledge This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on March 11, 2009 as DOI: 10.1124/mol.108.054395   a  t  A S P E T  J   o ur n a l   s  on S  e  p t   e m b  e r 1  9  ,2  0 1  6 m ol   ph  a r m . a  s  p e  t   j   o ur n a l   s  . or  gD o wnl   o a  d  e  d f  r  om 
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