Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer.
Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.
Herrick-Davis
Katharine
K
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA. daviskh@mail.amc.edu
Weaver
Barbara A
BA
Grinde
Ellinor
E
Mazurkiewicz
Joseph E
JE
D001426:Bacterial Proteins / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D044405:Receptor, Serotonin, 5-HT2C / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D044405:Receptor, Serotonin, 5-HT2C; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins