Research reportDifferential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors
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.
References (24)
- et al.
Beta-arrestin-biased agonism at the beta2-adrenergic receptor
J. Biol. Chem.
(2008) Ligand-selective receptor conformations revisited: the promise and the problem
Trends Pharmacol. Sci.
(2003)- et al.
Constitutively active mu-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin
J. Biol. Chem.
(2001) Agonist-induced regulation and trafficking of kappa opioid receptors
Life Sci.
(2004)- et al.
Structural plasticity associated with exposure to drugs of abuse
Neuropharmacology
(2004) - et al.
Basal opioid receptor activity, neutral antagonists, and therapeutic opportunities
Life Sci.
(2005) - et al.
Receptor-dependent RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement of Gq/G11
J. Biol. Chem.
(2003) - et al.
Differential G-protein activation by alkaloid and peptide opioid agonists in the human neuroblastoma cell line SK-N-BE
Biochem. J.
(1999) - et al.
Distinct differences between morphine- and [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin-mu-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation
J. Neurochem.
(1998) What's for lunch at the conformational cafeteria?
Mol. Pharmacol.
(2005)