Time-resolved fret between gpcr ligands reveals oligomers in native tissues

ABSTRACT G protein–coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as

no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling

with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these

transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two

protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary

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(2009). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS Thanks are due to S. Granier, P. Rondard and L. Prezeau for their critical reading of the manuscript. This work was

supported by research grants from the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Actions Concertées Incitatives “Molécules

Cibles et Thérapeutiques” (no. 240 and 355), ANR-06-BLAN-0087-03 and ANR-09-BLAN-0272) and the US National Institutes of Health (grants GM025280, DA022413, MH54137). This work was also made

possible by the Plateforme de Pharmacologie-Criblage of Montpellier and the Region Languedoc-Roussillon. NAPS and PPHT amines were synthesized by the National Institute of Mental Health

Chemical Synthesis and Drug Supply Program and provided by A.N. and J.J. AUTHOR INFORMATION Author notes * Laura Albizu & Michaela Kralikova Present address: Present addresses:

Department of Neurology, Mount Sinai School of Medicine, New York, New York, USA (L.A.); Department of Auditory Neuroscience, Czech Academy of Science, Prague, Czech Republic (M.K.)., *

Laura Albizu and Martin Cottet: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique,

Montpellier, France Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux * Institut National de la Santé et de la Recherche

Médicale, Montpellier, France Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux * Université Montpellier 1 and 2,

Montpellier, France Laura Albizu, Martin Cottet, René Seyer, Isabelle Brabet, Jean-Philippe Pin, Bernard Mouillac & Thierry Durroux * Center for Molecular Recognition, Columbia

University College of Physicians and Surgeons, New York, New York, USA Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch * Department of Psychiatry, Columbia University College of

Physicians and Surgeons, New York, New York, USA Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch * Division of Molecular Therapeutics, New York State Psychiatric Institute, New

York, New York, USA Michaela Kralikova, Marie-Laure Rives & Jonathan Javitch * Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, Ohio, USA

Stoytcho Stoev & Maurice Manning * Cisbio Bioassays, Bagnols sur Cèze, France Thomas Roux, Hervé Bazin, Emmanuel Bourrier, Laurent Lamarque & Eric Trinquet * Département de

Physiologie, Laboratoire de Neuroendocrinologie du Développement, Université des Sciences et Technologies de Lille, Villeneuve d′Ascq, France Christophe Breton * Medicinal Chemistry Section,

National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland, USA Amy Newman * Department of Pharmacology, Columbia University College

of Physicians and Surgeons, New York, New York, USA Jonathan Javitch Authors * Laura Albizu View author publications You can also search for this author inPubMed Google Scholar * Martin

Cottet View author publications You can also search for this author inPubMed Google Scholar * Michaela Kralikova View author publications You can also search for this author inPubMed Google

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this author inPubMed Google Scholar * Hervé Bazin View author publications You can also search for this author inPubMed Google Scholar * Emmanuel Bourrier View author publications You can

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publications You can also search for this author inPubMed Google Scholar * Marie-Laure Rives View author publications You can also search for this author inPubMed Google Scholar * Amy Newman

View author publications You can also search for this author inPubMed Google Scholar * Jonathan Javitch View author publications You can also search for this author inPubMed Google Scholar

* Eric Trinquet View author publications You can also search for this author inPubMed Google Scholar * Maurice Manning View author publications You can also search for this author inPubMed 

Google Scholar * Jean-Philippe Pin View author publications You can also search for this author inPubMed Google Scholar * Bernard Mouillac View author publications You can also search for

this author inPubMed Google Scholar * Thierry Durroux View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.A. and T.D. originated the project.

L.A., M.C. and T.D. executed most of the experiments and wrote the manuscript; S.S. and M.M. performed the peptide agonist synthesis; R.S. synthesized the antagonist peptide; I.B. performed

experiments with SNAP-tag receptors; M.-L.R., M.K., A.N. and J.J. contributed to the development of the fluorescent dopamine receptor ligands and assay system; T.R. characterized the

dopamine ligands; H.B., E.B. and L.L. labeled the ligands with fluorophores; C.B. performed saturation experiments with [125I]OTA; E.T., B.M. and J.-P.P. supported the project and

participated in the writing of the manuscript. CORRESPONDING AUTHORS Correspondence to Jean-Philippe Pin, Bernard Mouillac or Thierry Durroux. ETHICS DECLARATIONS COMPETING INTERESTS T.R.,

H.B., E.B., L.L.and E.T. are employees of Cisbio, which develops HTRF compatible fluorescent ligands and products combining its HTRF technology with Covalys SNAP-tag protein labeling system,

and therefore may gain financially through publication of this paper. Part of the work of CNRS UMR 5203 has been financially supported by Cisbio and by an unrestricted grant from Senomyx.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Tables 1–3, Supplementary Figures 1–3 and Supplementary Methods (PDF 2170 kb) RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Albizu, L., Cottet, M., Kralikova, M. _et al._ Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. _Nat Chem Biol_

6, 587–594 (2010). https://doi.org/10.1038/nchembio.396 Download citation * Received: 30 November 2009 * Accepted: 07 May 2010 * Published: 11 July 2010 * Issue Date: August 2010 * DOI:

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