RT Journal Article
SR Electronic
T1 Fluorescence Correlation Spectroscopy Analysis of Serotonin, Adrenergic, Muscarinic, and Dopamine Receptor Dimerization: The Oligomer Number Puzzle
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.113.087072
DO 10.1124/mol.113.087072
A1 Katharine Herrick-Davis
A1 Ellinor Grinde
A1 Ann Cowan
A1 Joseph E. Mazurkiewicz
YR 2013
UL http://molpharm.aspetjournals.org/content/early/2013/08/01/mol.113.087072.abstract
AB The issue of G-protein coupled receptor (GPCR) oligomer status has not been resolved. While many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCR. Previous studies have reported monomers, dimers, tetramers and higher order oligomers. In the present study this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCR were selected from the serotonin (5-HT2A), adrenergic (α1b-AR and β2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with GFP or YFP and expressed in HEK293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5x10-9 cm2/s. PCH molecular brightness analysis was used to determine GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCR revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced chi square analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.