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Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies.

ABSTRACT: The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially recognize activated receptors. Recently, we generated monoclonal antibodies that recognize regions proximal to glycosylation sites in the receptor N terminus. Characterization of these antibodies revealed that agonist treatment leads to a decrease in epitope recognition by the antibody presumably because of a movement of the region of the N terminus proximal to glycosylation sites. The time course of the decrease in antibody recognition suggested that it could be due to a post-activation-mediated event. Examination of the involvement of receptor residues in the C-tail and beta-arrestin binding using site-directed mutagenesis and cells or tissues lacking beta-arrestin 2 suggests a role for these desensitization-related mechanisms in governing antibody binding to the receptor. Thus, these N-terminally directed antibodies can differentially recognize post-activation-mediated changes in the C-terminal (intracellular) region of the receptor. Therefore, these conformation-sensitive antibodies represent powerful reagents to probe receptor activation states and provide a potential tool for identifying and characterizing new compounds of therapeutic interest.

SUBMITTER: Gupta A

PROVIDER: S-EPMC2447646 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies.

Gupta Achla A Rozenfeld Raphael R Gomes Ivone I Raehal Kirsten M KM Décaillot Fabien M FM Bohn Laura M LM Devi Lakshmi A LA

The Journal of biological chemistry 20080206 16

The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially rec ...[more]

PMID: 18256033

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Project description:Although participation of opioids in antinociception induced by cannabinoids has been documented, there is little information regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids could be involved in peripheral antinociception induced by activation of mu-, delta- and kappa-opioid receptors.Nociceptive thresholds to mechanical stimulation of rat paws treated with intraplantar prostaglandin E2 (PGE2, 2 microg) to induce hyperalgesia were measured 3 h after injection using an algesimetric apparatus. Opioid agonists morphine (200 microg), (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) (80 microg), bremazocine (50 microg); cannabinoid receptor antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (20-80 microg), 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl) methanone (AM630) (12.5-100 microg); and an inhibitor of methyl arachidonyl fluorophosphonate (MAFP) (1-4 microg) were also injected in the paw.The CB1-selective cannabinoid receptor antagonist AM251 completely reversed the peripheral antinociception induced by morphine in a dose-dependent manner. In contrast, the CB2-selective cannabinoid receptor antagonist AM630 elicited partial antagonism of this effect. In addition, the administration of the fatty acid amide hydrolase inhibitor, MAFP, enhanced the antinociception induced by morphine. The cannabinoid receptor antagonists AM251 and AM630 did not modify the antinociceptive effect of SNC80 or bremazocine. The antagonists alone did not cause any hyperalgesic or antinociceptive effect.Our results provide evidence for the involvement of endocannabinoids, in the peripheral antinociception induced by the mu-opioid receptor agonist morphine. The release of cannabinoids appears not to be involved in the peripheral antinociceptive effect induced by kappa- and delta-opioid receptor agonists.

Project description:Background and purposeAlthough opioids have been reported to affect glucose homeostasis, relatively little is known on the role of ?-opioid receptors. We have investigated the regulation of glucose transport by human ?-opioid receptors expressed in Chinese hamster ovary cells.Experimental approachThe uptake of [(3)H]-2-deoxy-D-glucose and 3-O-[methyl-[(3)H]]-D-glucose in response to ?-opioid receptor ligands and the expression of GLUT1, GLUT3 and GLUT4 glucose transporters were examined. Moreover, the effects of intracellular signal transduction inhibitors on ?-opioid receptor-regulated [(3)H]-2-deoxy-D-glucose uptake and protein phosphorylation were investigated.Key resultsActivation of ?-opioid receptors rapidly stimulated [(3)H]-2-deoxy-D-glucose and 3-O-[methyl-[(3)H]]-D-glucose uptakes, which were blocked by the GLUT inhibitors cytochalasin B and phloretin. The stimulation of [(3)H]-2-deoxy-D-glucose uptake that occurred without a change in plasma membrane GLUT1 - required the coupling to G(i) /G(o) proteins - was independent of cAMP and extracellular signal-regulated protein kinases, and was suppressed by blockade of Src and insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinases. Inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin or LY294002 and by PI3K?, but not ?, isoform-selective inhibitors greatly reduced the ?-opioid receptor stimulation of glucose uptake. Moreover, the response was attenuated by overexpressing a dominant-negative kinase-deficient Akt form and by chemical inhibition of Akt. Stimulation of ?-opioid receptors increased protein kinase C?/? (PKC?/?) phosphorylation and a selective PKC?/? inhibitor slightly reduced opioid stimulation of glucose uptake.Conclusions and implications?-Opioid receptors stimulated glucose transport probably by enhancing GLUT1 intrinsic activity through a signalling cascade involving G(i)/G(o), Src, IGF-1R, PI3K?, Akt and, to a minor extent, PKC?/?. This effect may contribute to the opioid regulation of glucose homeostasis in physio-pathological conditions.

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Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies.

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Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies.

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