Evidence for Distinct Antagonist-Revealed Functional States
of 5-Hydroxytryptamine
2A
Receptor Homodimers
□ S
Jose ´ Brea, Maria ´ n Castro, Jesu ´ s Giraldo, Juan F. Lo ´ pez-Gime ´ nez, Juan Fernando Padín,
Fa ´ tima Quintia ´ n, Maria Isabel Cadavid, Maria Teresa Vilaro ´ , 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., M.I.L.); Institut de Neurocie ` ncies and Unitat de
Bioestadística, Universitat Auto ` noma de Barcelona, Bellaterra, Spain (J.G.); Department of Biochemistry and Molecular Biology,
University of Glasgow, Glasgow, United Kingdom (J.F.L.-G., G.M.); Department of Neurochemistry and Neuropharmacology,
Instituto de Investigaciones Biome ´ dicas de Barcelona, Consejo Superior de Investigaciones Cientificas, IDIBAPS, Barcelona,
Spain (M.T.V., G.M.); Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas (W.P.C.,
K.A.B.); Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
(J.-P.V.); and Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, Boston Massachusetts (J.-P.V.)
Received December 23, 2008; accepted March 11, 2009
ABSTRACT
The serotonin (5-hydroxytryptamine; 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 fluores-
cence resonance energy transfer and immunoprecipitation
studies that the 5-HT
2A
-receptor homodimerized 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 haloper-
idol, 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.
G protein-coupled receptors (GPCRs) constitute the major
family of cell surface proteins involved in cell signaling cas-
cades 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 li-
gand-GPCR-effector complexes to fine-tune their own sig-
nals, broadening its apparent simplicity and highlighting
ligands as individual chemical species capable of transmit-
ting messages into cellular function with a versatility unpre-
dicted 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 con-
formations 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 accumu-
lated experimental evidence has led to the development of
novel mathematical representations that attempt to explain
the chemical biology of GPCRs and integrate the new knowl-
This work was supported by the Ministerio de Educacio ´n y Ciencia, Spain
[Grants SAF2007-65913, SAF2005-08025-C03]; the Xunta de Galicia [Grant
PGIDIT06PXID203186PR, 2007/118]; and by Red Tema ´ tica de Investigacio ´n
Cooperativa COMBIOMED from Instituto de Salud Carlos III and Fundacio ´
La Marato ´ de TV3 [Reference 070530].
J.B., M.C., and J.G. contributed equally to this work.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.108.054395.
□ S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine, serotonin; AA, arachidonic acid; CHO, Chinese hamster ovary;
()DOB, ()-1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane; ()DOI, ()-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; FCS, fetal calf se-
rum; GR55562, (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenylbenzamide dihydrobromide]; IP, inositol phosphate; MDL100,907,
(R)-()-4-[1-hydroxy-1(2,3-dimethoxyphenyl)metyhyl]N-2– 4-fluorophenylethyl)piperidine; PLA
2
, phospholipase A
2
; PLC, phospholipase C; HEK,
human embryonic kidney; MEM, minimum essential medium; PCR, polymerase chain reaction; CFP, cyan fluorescent protein; YFP, yellow
fluorescent protein; FRET, fluorescence resonance energy transfer; RIPA, radioimmunoprecipitation assay; PAGE, polyacrylamide gel electro-
phoresis; TTBS, Tris-buffered saline/Tween 20; compound 40/80, p-methoxy-N-methyl-phenethylamine.
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Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 54395/3472926
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