title: Regulation of oligomeric organization of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor observed by spatial intensity distribution analysis.
abstract: The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: John D
authors.initials: JD
authors.lastname: Pediani
authors.affiliation: the University of Bordeaux, LP2N, UMR 5298, F-33405 Talence, France, and the Institut d'Optique Graduate School and CNRS, LP2N, UMR 5298, F-33405 Talence, France.
authors.forename: Antoine G
authors.initials: AG
authors.lastname: Godin
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, Graeme.Milligan@glasgow.ac.uk.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology*; D020836:Protein Structure, Quaternary; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000494:pharmacology
chemical_list: D046912:Multiprotein Complexes; D044405:Receptor, Serotonin, 5-HT2C; D011993:Recombinant Fusion Proteins; D058830:Serotonin 5-HT2 Receptor Antagonists; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: G protein-coupled receptor (GPCR); epidermal growth factor receptor (EGFR); serotonin; seven-helix receptor; single-molecule biophysics
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