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J. Biol. Chem., Vol. 280, Issue 44, 37225-37235, November 4, 2005

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Dopamine Receptor Oligomerization Visualized in Living Cells^*

From the Departments of Pharmacology and Medicine, University of Toronto, Toronto, Ontario M5S 1A8 and Centre for Addiction and Mental Health, Toronto, Ontario M5T 1R8, Canada

G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.

Received for publication, April 26, 2005 , and in revised form, July 8, 2005.

^* This work was supported by a Proof of Principle Grant from the Canadian Institutes for Health Research. 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.

² Holder of a Canada Research Chair in Molecular Neuroscience.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Medical Science Bldg., University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-7579; Fax: 416-978-2733; E-mail: brian.odowd{at}utoronto.ca.

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