title: Native serotonin 5-HT2C receptors are expressed as homodimers on the apical surface of choroid plexus epithelial cells.
abstract: G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/μm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.) daviskh@mail.amc.edu.
authors.forename: Katharine
authors.initials: K
authors.lastname: Herrick-Davis
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Ellinor
authors.initials: E
authors.lastname: Grinde
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Tara
authors.initials: T
authors.lastname: Lindsley
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Milt
authors.initials: M
authors.lastname: Teitler
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Filippo
authors.initials: F
authors.lastname: Mancia
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Ann
authors.initials: A
authors.lastname: Cowan
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York (K.H.-D., E.G., T.L., M.T., J.E.M.); Department of Physiology and Cellular Biophysics, Columbia University, New York, New York (F.M.); and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut (A.C.).
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Mazurkiewicz
mesh_terms: D000345:Affinity Labels; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D011869:Radioligand Assay; D017207:Rats, Sprague-Dawley; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D044405:Receptor, Serotonin, 5-HT2C; D049452:Green Fluorescent Proteins