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Morphine-like opiates selectively antagonize receptor-arrestin interactions.

ABSTRACT: The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

SUBMITTER: Molinari P

PROVIDER: S-EPMC2857100 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Morphine-like opiates selectively antagonize receptor-arrestin interactions.

Molinari Paola P Vezzi Vanessa V Sbraccia Maria M Grò Cristina C Riitano Daniela D Ambrosio Caterina C Casella Ida I Costa Tommaso T

The Journal of biological chemistry 20100226 17

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affecte ...[more]

PMID: 20189994

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Project description:PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Dataset Information

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

Publications

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

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