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J. Biol. Chem., Vol. 281, Issue 37, 27109-27116, September 15, 2006

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Serotonin 5-HT_2C Receptor Homodimer Biogenesis in the Endoplasmic Reticulum

REAL-TIME VISUALIZATION WITH CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER^*

From the Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208

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-HT_2C 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-HT_2C 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-HT_2C 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-HT_2C receptor maturation and processing.

Received for publication, May 8, 2006 , and in revised form, July 18, 2006.

^* This work was supported by National Institutes of Health Grants MH057019 (to K. H.-D.) and RR017926 (to J. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Neuropharmacology and Neuroscience, MC-136, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-6357; Fax: 518-262-5799; E-mail: daviskh{at}mail.amc.edu.

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