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Serotonin 5‐HT2C receptor homodimers identified by Fluorescence Correlation Spectroscopy
Author(s) -
Herrick-Davis Katharine,
Grinde Ellinor,
Mazurkiewicz Joseph E
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.663.6
Subject(s) - fluorescence correlation spectroscopy , receptor , yellow fluorescent protein , chemistry , fluorescence , bimolecular fluorescence complementation , biophysics , green fluorescent protein , biochemistry , biology , molecule , physics , organic chemistry , quantum mechanics , gene , yeast
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) were applied to determine diffusion rate and oligomeric size of G‐protein‐coupled receptors at the single molecule level. FCS was used to monitor fluctuations in fluorescence intensity arising from fluorescent‐tagged serotonin 5‐ HT 2C receptors freely diffusing within the plasma membrane of HEK293 cells and primary hippocampal neurons. FCS reported diffusion rates of 0.9μm 2 /s and 1.3μm 2 /s for GFP‐ and YFP‐tagged receptors, respectively. Since the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins within a cluster it can be used to determine the oligomer state of a protein complex. PCH analysis of fluorescent‐tagged 5‐HT 2C receptors provided molecular brightness values that were approximately twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5‐HT. Bimolecular fluorescence complementation (BiFC) of the N‐ and C‐terminal halves of YFP, attached to 5‐HT 2C receptors, was observed on the plasma membrane with a brightness equal to the monomeric YFP control. Co‐expression of GFP‐tagged 5‐HT 2C receptors with a large excess of non‐tagged 5‐HT 2C receptors decreased the brightness by half. These results indicate that the mobile fraction of 5‐HT 2C receptors in the plasma membrane is dimeric. Supported by NIH‐ MH86796.