Molecular and Cellular Pharmacology Separation and reformation of cell surface dopamine receptor oligomers visualized in cells ☆ Brian F. O'Dowd a,b, ⁎, Xiaodong Ji a,b , Mohammad Alijaniaram a,b , Tuan Nguyen a,b , Susan R. George a,b,c a Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada M5T IR8 b Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 c Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8 abstract article info Article history: Received 9 October 2010 Received in revised form 31 January 2011 Accepted 17 February 2011 Available online 1 March 2011 Keywords: G protein coupled receptor Dopamine receptor Nuclear localization Protein structure Oligomer We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR–NLS is retained at the cell surface, and following ligand removal, the GPCR–NLS translocated from the cell surface. The D 1 dopamine receptor expressed with either D 2 –NLS or D 1– NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D 1 –D 2 dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D 1 receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D 1 –D 2 heteromer. Dopamine D 1 or D 2 receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D 1 –D 2 heterooligomer contains D 1 homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation. © 2011 Elsevier B.V. All rights reserved. 1. Introduction G protein coupled receptors (GPCRs) form oligomers (Bouvier, 2001; McVey et al., 2001; George et al., 2002; O'Dowd et al., 2005), and arrays of oligomers (Liang et al., 2003; Fotiadis et al., 2003). GPCRs also form heterooligomers (Jordan and Devi, 1999; George et al., 2000; Milligan, 2004). However many details of oligomer formation remain unknown. We reported that dopamine receptor D 1 –D 2 heterooligomers existed in brain (Lee et al., 2004), and showed these receptors existed within a heterooligomer (So et al., 2005), in cultured striatal neurons and brain (Hasbi et al., 2009; Perreault et al., 2010). We showed synergism of receptor activation within D 1 –D 2 heteromers generating a Gq-mediated calcium signal (Lee et al., 2004; Rashid et al., 2007; Hasbi et al., 2009). The D 1 –D 2 heterooligomer displayed agonist- induced co-internalization and trafficking (So et al., 2005). Within this heterooligomer, the individual receptors were altered (Rashid et al., 2007). GPCR oligomerization occurs in the endoplasmic reticulum (Hasbi et al., 2007), and are subjected to quality control before exiting to the cell surface (Kong et al., 2007), and oligomers internalize as oligomers after agonist exposure (So et al., 2007). We question whether GPCR oligomers are dynamically regulated in response to ligand activation. In addition to the biosynthesis of GPCR oligomers in the ER, oligomers may form at the cell surface (Law et al., 2005) and be broken apart (Lambert, 2010). Although we have used cell surface time-resolved FRET (So et al., 2005), confocal FRET (Hasbi et al., 2009) and BRET (Hasbi et al., 2007), to determine receptor:receptor interactions, these methods are limited when investigating dynamic oligomer regulation. The unanswered question was the fate of GPCR oligomers following agonist activation. The following need to be resolved: (i) whether constituent receptors of hetero- or homooligomers remain together or separate following agonist treatment, (ii) whether the separated constituents reform into a heterooligomer at the cell surface, and (iii) if GPCR homooligomers and heterooligomers were regulated differently following agonist treatment. We developed a method to visualize regulation of oligomers (O'Dowd et al., 2005). A nuclear localization sequence (NLS) was European Journal of Pharmacology 658 (2011) 74–83 ☆ This work was partially supported by a Proof of Principle Grant from the Canadian Institutes for Health Research. SRG holds a Canada Research Chair in Molecular Neuroscience. ⁎ Corresponding author at: Department of Pharmacology, University of Toronto, 1 King's College Circle, Room 4353, Toronto, Ontario, Canada M5S 1A8. Tel.: + 1 416 978 7579; fax: +1 416 971 2868. E-mail address: brian.odowd@utoronto.ca (B.F. O'Dowd). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.02.030 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar