Ligand-Induced Regulation and Localization of Cannabinoid CB1 and Dopamine D2L Receptor Heterodimers □S

The cannabinoid CB1 (CB1) and dopamine D2 (D2) receptors are coexpressed in the basal ganglia, an area of the brain involved in such processes as cognition, motor function, and emotional control. Several lines of evidence suggest that CB1 and D2 receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB1 and D2 receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB1-D2L heterodimers as well as D2L-D2L homodimers in a neuronal cell model, Cath. a differentiated cells. MBiFC was then used to explore the effects of persistent ligand treatment on receptor dimerization at the plasma membrane and intracellularly. Persistent (20-h) agonist treatment resulted in increased formation of CB1-D2L heterodimers relative to the D2L-D2L homodimers. The effects of the D2 agonist quinpirole were restricted to the intracellular compartment and may reflect increased D2L receptor expression. In contrast, treatment with the CB1 receptor agonist (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55, 940) produced increases in both membrane and intracellular CB1D2L heterodimers independently of alterations in CB1 receptor expression. The effects of CB1 receptor activation were attenuated by the CB1 antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both timeand dose-dependent. The effects of CB1 activation were examined further by combining MBiFC with a constitutively active CB1 receptor mutant, CB1T210I. These studies demonstrated that the expression of CB1T210I increased intracellular CB1-D2L heterodimer formation. In summary, agonist-induced modulation of CB1-D2L oligomerization may have physiological implications in diseases such as Parkinson’s disease and drug abuse. Increasing evidence suggests that G protein-coupled receptors (GPCRs) may function in receptor dimeric or higher order oligomeric complexes (for review, see Milligan, 2008). One set of receptors that has received significant attention relevant to oligomerization is the CB1 cannabinoid (CB1) receptor and dopamine D2 (D2) receptor (for review, see Fuxe et al., 2008). It is thought that the cannabinoid system negatively modulates dopamine circuits as activation of the CB1 receptor leads to an attenuation of dopamine signaling (Laviolette and Grace, 2006). The CB1 receptor is widely expressed in the central nervous system, with great abundance in the basal ganglia (Herkenham et al., 1991). CB1 receptors are located on striatal GABAergic neurons (Herkenham et al., 1991), and they are also found on dendrites in both the dorsal striatum and the nucleus accumbens (Pickel et al., 2006). The D2 receptor exists as two splice variants, D2S (short) and D2L (long). The D2S variant is highly expressed on presynaptic dopaminergic neurons, whereas the D2L variant is found postsynaptically on dopaminergic neurons throughout the striatum This work was supported by the National Institutes of Health National Institute of Mental Health [Grant MH060397]; and the Purdue Research Foundation and the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.162701. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: GPCR, G protein-coupled receptor; CB1, cannabinoid 1 receptor; CC, Cerulean C-terminal fragment; D2, dopamine D2 receptor; D2S, short form of D2 receptor; D2L, long form of D2 receptor; FRET, fluorescence resonance energy transfer; BRET, bioluminescence resonance energy transfer; BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; MBiFC, multicolor bimolecular fluorescence complementation; CAD, Cath. a differentiated; CP55,940, (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl) cyclohexanol; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; YFP, yellow fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; CFP, cyan fluorescent protein; VN, Venus N-terminal fragment; CN, Cerulean N-terminal fragment; CC, C-terminal fragment of Cerulean; cFRET, corrected fluorescence resonance energy transfer; M, muscarinic receptor; A2A adenosine2A receptor; AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4morpholinyl-1H-pyrazole-3-carboxamide; ER, endoplasmic reticulum; wt, wild type. 0022-3565/10/3323-710–719$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 162701/3563778 JPET 332:710–719, 2010 Printed in U.S.A. 710 http://jpet.aspetjournals.org/content/suppl/2009/12/17/jpet.109.162701.DC1 Supplemental material to this article can be found at: at A PE T Jornals on July 0, 2017 jpet.asjournals.org D ow nladed from (Khan et al., 1998; Usiello et al., 2000). These observations reveal that CB1 and D2L receptors have overlapping expression patterns in the striatum and also suggest that they are colocalized in neurons in the nucleus accumbens (see references within Kearn et al., 2005; Pickel et al.,