Abstract
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 CB1-D2L 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)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both time- and 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.
Footnotes
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.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
↵1 Personal communications confirmed that the D2L receptor was used in Glass and Felder (1997) (per Dr. David Sibley, who supplied the CHO-D2 cells) as well as in Marcellino et al. (2008) (per Dr. Kjell Fuxe).
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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-4-morpholinyl-1H-pyrazole-3-carboxamide
- ER
- endoplasmic reticulum
- wt
- wild type.
- Received November 2, 2009.
- Accepted December 15, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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