title: Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers.
abstract: Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA. daviskh@mail.amc.edu
authors.forename: Katharine
authors.initials: K
authors.lastname: Herrick-Davis
authors.forename: Ellinor
authors.initials: E
authors.lastname: Grinde
authors.forename: Tara
authors.initials: T
authors.lastname: Lindsley
authors.forename: Ann
authors.initials: A
authors.lastname: Cowan
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Mazurkiewicz
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004058:Diffusion / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005453:Fluorescence; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055503:Protein Multimerization; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D044405:Receptor, Serotonin, 5-HT2C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D044405:Receptor, Serotonin, 5-HT2C; D049452:Green Fluorescent Proteins; D012701:Serotonin