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Distinct roles of ?-arrestin 1 and ?-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1).

ABSTRACT: The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070-12082). Here, we identify the mediators of CB1 receptor internalization and ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an ORG27569-induced signaling mechanism for CB1 wherein ?-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-?-arrestin 1 upon ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of ?-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with ORG27569. These results demonstrate for the first time the involvement of ?-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two ?-arrestin isoforms in CB1 signaling and internalization.

SUBMITTER: Ahn KH

PROVIDER: S-EPMC3617280 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Distinct roles of β-arrestin 1 and β-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1).

Ahn Kwang H KH Mahmoud Mariam M MM Shim Joong-Youn JY Kendall Debra A DA

The Journal of biological chemistry 20130228 14

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy ...[more]

PMID: 23449980

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Project description:The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and ?(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.

Dataset Information

Distinct roles of ?-arrestin 1 and ?-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1).

Publications

Distinct roles of β-arrestin 1 and β-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1).

Similar Datasets

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