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Ligand-induced regulation and localization of cannabinoid CB1 and dopamine D2L receptor heterodimers.

ABSTRACT: The cannabinoid CB(1) (CB(1)) and dopamine D(2) (D(2)) 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 CB(1) and D(2) receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB(1) and D(2) receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB(1)-D(2L) heterodimers as well as D(2L)-D(2L) 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 CB(1)-D(2L) heterodimers relative to the D(2L)-D(2L) homodimers. The effects of the D(2) agonist quinpirole were restricted to the intracellular compartment and may reflect increased D(2L) receptor expression. In contrast, treatment with the CB(1) 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 CB(1)-D(2L) heterodimers independently of alterations in CB(1) receptor expression. The effects of CB(1) receptor activation were attenuated by the CB(1) 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 CB(1) activation were examined further by combining MBiFC with a constitutively active CB(1) receptor mutant, CB(1)T210I. These studies demonstrated that the expression of CB(1)T210I increased intracellular CB(1)-D(2L) heterodimer formation. In summary, agonist-induced modulation of CB(1)-D(2L) oligomerization may have physiological implications in diseases such as Parkinson's disease and drug abuse.

SUBMITTER: Przybyla JA

PROVIDER: S-EPMC2835427 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Ligand-induced regulation and localization of cannabinoid CB1 and dopamine D2L receptor heterodimers.

Przybyla Julie A JA Watts Val J VJ

The Journal of pharmacology and experimental therapeutics 20091216 3

The cannabinoid CB(1) (CB(1)) and dopamine D(2) (D(2)) 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 CB(1) and D(2) receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB(1) and D(2) receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complemen ...[more]

PMID: 20016021

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Project description:Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTP?S (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTP?S binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.

Project description:ObjectivesWe aimed to explore the effects of pharmacologic inhibition of cannabinoid-1 (CB1) receptor in in vivo and in vitro models of doxorubicin (DOX)-induced cardiotoxicity.BackgroundDoxorubicin is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Endocannabinoids mediate cardiodepressive effects through CB1 receptors in various pathophysiological conditions, and these effects can be reversed by CB1 antagonists.MethodsLeft ventricular function was measured by Millar pressure-volume system. Apoptosis markers, CB1/CB2 receptor expression, and endocannabinoid levels were determined by immunohistochemistry, Western blot, reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, flow cytometry, fluorescent microscopy, and liquid chromatography/in-line mass spectrometry techniques.ResultsFive days after the administration of a single dose of DOX (20 mg/kg intraperitoneally) to mice, left ventricular systolic pressure, maximum first derivative of ventricular pressure with respect to time (+dP/dt), stroke work, ejection fraction, cardiac output, and load-independent indexes of contractility (end-systolic pressure-volume relation, preload-recruitable stroke work, dP/dt-end-diastolic volume relation) were significantly depressed, and the myocardial level of the endocannabinoid anandamide (but not CB1/CB2 receptor expression) was elevated compared with vehicle-treated control mice. Treatment with the CB1 antagonists rimonabant or AM281 markedly improved cardiac dysfunction and reduced DOX-induced apoptosis in the myocardium. Doxorubicin also decreased cell viability and induced apoptosis in the H9c2 myocardial cell line measured by flow cytometry and fluorescent microscopy, which were prevented by the preincubation of the cells with either CB1 antagonist, but not with CB1 and CB2 agonists and CB2 antagonists.ConclusionsThese data suggest that CB1 antagonists may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

Dataset Information

Ligand-induced regulation and localization of cannabinoid CB1 and dopamine D2L receptor heterodimers.

Publications

Ligand-induced regulation and localization of cannabinoid CB1 and dopamine D2L receptor heterodimers.

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