Elsevier

Brain Research Bulletin

Volume 77, Issue 1, 5 September 2008, Pages 49-54
Brain Research Bulletin

Research report
Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors

https://doi.org/10.1016/j.brainresbull.2008.05.003Get rights and content

Abstract

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such “agonist-directed” signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Gα12 and that both morphine and DAMGO decreased Gαi3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20 h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Gα12 and Gαi3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Gα12 expression. All five mu agonists decreased Gαi3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Gαi3 expression or moderately up-regulated Gα12 expression. Among five kappa agonists, ((−)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (−)-U50,488) tested with hKOR-CHO cells, only chronic (−)-U50,488 and (−)-EKC up-regulated Gα12 expression. All kappa agonists decreased Gαi3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling.

Introduction

As postulated by Kenakin [8], [9], all seven transmembrane receptors such as β2-adrenergic receptors [5], are likely capable of adopting a range of distinct conformations, each of which can lead to the activation of distinct intracellular signaling pathways. The adoption of these distinct conformations can, in turn, be modulated by the presence of ligand. This ligand-related behavior has been termed “agonist-directed trafficking”, “stimulus trafficking”, “functional selectivity” and “biased agonism” [3], [17]. Ligand-directed signaling to different cellular effector pathways extends drug effects from ligand-selective receptor conformations to the relationships between those conformations, cellular function, and ultimately therapeutics. Thus, it is possible to develop response-selective drugs that maximize therapeutic efficacy and minimize unwanted effects.

A growing body of evidence indicates that opioid receptor ligands demonstrate biased agonism. For example, Allouche et al. [1] showed that δ peptide agonists and the non-peptide ligand, etorphine, activate different G protein subunits. Photolabeling of Gα-subunits with azidoanilido-[α-32P]GTP showed that constitutively active μ opioid receptors activate individual G proteins differently than those stimulated by DAMGO (Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol) [12] and that peptide and non-peptide μ agonists induce different patterns of μ receptor phosphorylation [2]. Moreover, opioid agonists differ in their ability to induce receptor internalization of μ [10], δ [11] and κ [13] opioid receptors. In this regard, recent studies showed that herkinorin (HERK) ((2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester) is a non-nitrogenous neoclerodane diterpene fully efficacious μ agonist [7], that unlike DAMGO, does not promote β-arrestin recruitment and μ receptor internalization, even in cells that over express β-arrestin and the GPCR kinase, GRK2 [6]. Other data indicated that although morphine does not promote β-arrestin recruitment and μ receptor internalization in CHO cells, it does promote μ receptor internalization in cells that over express β-arrestin and the GRK2 [19].

We previously reported that chronic treatment of CHO cells expressing the cloned human μ opioid receptor (hMOR-CHO) with morphine decreased the expression of Gαi2 (64%) and Gαi3 (60%), had no effect on Gαo expression, and, unexpectedly, increased Gα12 expression (66%). These changes did not occur in cells expressing a mutant μ opioid receptor (T394A-CHO) that do not develop morphine tolerance and dependence, indicating that morphine-induced changes in the expression of these G protein subunits are related to the development of tolerance and dependence [24].

With interest focused on Gαi3 and Gα12, more recent work supported the occurrence of biased agonism in hMOR-CHO cells treated chronically with different μ agonists [22]. Of direct relevance to the present study, we demonstrated that after chronic (20 h) treatment of hMOR-CHO cells with DAMGO, morphine or HERK, only chronic morphine increased the expression level of Gα12 [24], a G protein that regulates downstream cytoskeletal proteins via its control of RhoA [4], [18]. Numerous studies have documented that the small GTPase RhoA is involved in the regulation of various cellular functions such as remodeling of the actin cytoskeleton and induction of transcriptional activity. Gα12/Gα13 are the major upstream regulators of RhoA activity. The thrombin receptor PAR-1 has been shown to couple to all three G protein families (Gα12/Gα13, Gαq and Gαi) and to regulate a substantial network of signaling pathways [4].

The ability of chronic morphine, but not chronic HERK or DAMGO, to up-regulate Gα12 expression in hMOR-CHO cells, is an example of biased agonism. Thus, in the present study we further tested the hypothesis that chronic opioid-agonist regulation of G protein expression is agonist-directed using CHO cells that express the cloned human μ, δ or κ opioid receptors. Following a 20 h treatment of cells with chemically distinct opioid agonists, our data demonstrate that chronic opioid agonist regulation of G protein expression depends on the agonist tested, providing additional evidence for biased agonism.

Section snippets

Cell culture and crude membrane preparation

The recombinant CHO cells (hMOR-CHO, hDOR-CHO, hKOR-CHO) were produced by stable transfection with the human opioid receptor cDNA, and provided by Toll et al. [16]. The cells were grown on plastic flasks in DMEM/F-12 (50%/50%) medium (hMOR-CHO) or in DMEM medium (hDOR-CHO and hKOR-CHO) containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and G-418 (0.20–0.25 mg/ml) under 95% air/5% CO2 at 37 °C. We used a modified cell culture medium for experiments involving western blots for Gα12.

Initial experiments

The initial set of experiments was designed to determine the EC50 values of the test agents in the functional [35S]-GTP-γ-S binding assay (Table 1). In subsequent experiments, cells were then treated for 20 h with drug concentrations approximately 100–150 times the corresponding EC50 values in the [35S]-GTP-γ-S binding assay. The next series of experiments was conducted to further verify that the conditions of the chronic drug treatment used in this study produced opioid tolerance, as assessed

Discussion

The occurrence of biased agonism is well established for many GPCRs [8], [3]. However, the mechanisms and functional consequences of agonist-directed signaling remain to be clarified. Because of the role that receptor internalization plays in opioid receptor desensitization and opioid tolerance, studies of biased agonism, with the end-point of receptor internalization, is a subject of contemporary interest. Opioid agonists, for example, differ in their ability to induce receptor internalization

Conflict of Interest

The authors have no conflict of interest.

Acknowledgements

This research was supported by the Intramural Research Program of the NIH, National Institute on Drug Abuse and funding NIDA grant DA018151 to Dr. Prisinzano.

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