Article
- The EMBO Journal (2008) 27, 2293 - 2304
- doi:10.1038/emboj.2008.153
Published online: 31 July 2008
Subject Category:
Dopamine D2 receptors form higher order oligomers at physiological expression levels
Wen Guo1,2,a, Eneko Urizar1,2,a, Michaela Kralikova1,2, Juan Carlos Mobarec4, Lei Shi5, Marta Filizola4 and Jonathan A Javitch1,2,3
- Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY, USA
- Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY, USA
- Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA
- Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA
- Department of Physiology and Biophysics and the Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY, USA
Correspondence to:
Jonathan A Javitch, Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, College of Physicians and Surgeons, Columbia University, 630W 168th street, P&S, Room 11-401, NY 10032, USA. Tel.: +1 21 230 573 08; Fax: +1 21 230 555 94; E-mail: jaj2@columbia.edu
aThese authors contributed equally to this work
Received 27 February 2008; Accepted 9 July 2008
Abstract
G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.
Keywords:
- BRET,
- crosslinking,
- dopamine,
- GPCR,
- higher order
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