Functional selectivity at the μ-opioid receptor: implications for understanding opioid analgesia and tolerance

Abstract

Opioids are the most effective analgesic drugs for the management of moderate or severe pain, yet their clinical use is often limited because of the onset of adverse side effects. Drugs in this class produce most of their physiological effects through activation of the μ opioid receptor; however, an increasing number of studies demonstrate that different opioids, while presumably acting at this single receptor, can activate distinct downstream responses, a phenomenon termed functional selectivity. Functional selectivity of receptor-mediated events can manifest as a function of the drug used, the cellular or neuronal environment examined, or the signaling or behavioral measure recorded. This review summarizes both in vitro and in vivo work demonstrating functional selectivity at the μ opioid receptor in terms of G protein coupling, receptor phosphorylation, interactions with β-arrestins, receptor desensitization, internalization and signaling, and details on how these differences may relate to the progression of analgesic tolerance after their extended use.

Fig. 1.

Schematic demonstrating key points in opioid receptor signaling and regulation that have been shown to be influenced by differential agonist occupation. A, heterotrimeric G proteins represent 16 individual gene products for Gα, 5 individual gene products for Gβ and 11 for Gγ proteins. Together, the diversity arising from heterotrimeric G protein subunit composition presents a gateway to potentially high diversification of agonist-directed coupling between μOR and G proteins. These interactions can determine access to secondary cascade activation. B, the μOR can be phosphorylated in response to agonist occupation by multiple kinases, each of which has multiple isoforms. Phosphorylation by a particular kinase may dictate secondary cascade interactions or subsequent receptor fate. CKII, casein kinase II. C, receptor interaction with scaffolding partners such as β-arrestins can be dependent or independent of receptor phosphorylation. Agonist occupancy may determine these interactions with potential binding partners. Such interactions can prevent (desensitization) or promote subsequent signaling. D, the μOR can be internalized in response to agonist occupancy. Endocytosis may involve clathrin- or caveolin-dependent processes and may result in the activation of subsequent signaling pathways, receptor recycling or degradation.

Fig. 2.

Schematic representation of functional selectivity at the level of GPCR and G protein or β-arrestin bias. In this diagram, agonists 1 and 2 both activate receptor G protein-coupling pathways, represented as “Signaling Pathway A.” However, agonist 1 leads to recruitment of β-arrestin but agonist 2 does not. Receptor-β-arrestin interactions can serve to disrupt receptor-G protein coupling and may also serve to facilitate GPCR signaling to alternate pathways such as Signaling Pathway B (Schmid and Bohn, 2010). By not recruiting β-arrestins, agonist 2 activates the receptor without engaging signaling pathway B while allowing signaling pathway A to proceed. Agonist 2 would be considered biased against β-arrestin signaling.

Fig. 3.

μOR phosphorylation in response to diverse opioid ligands. Radiolabeling of phosphorylated receptors was performed according to the methods described in detail by Oakley et al. (1999). HEK-293 cells expressing HA-μOR 1 were incubated in phosphate-free media in the presence of [³²P]orthophosphate (100 μCi/ml) for 1 h at 37°C. Opioid agonists were included at the doses indicated for 15 min at 37°C. Cells were lysed, and equivalent levels of receptor per sample (as calculated by simultaneous radioligand binding assays) were immunoprecipitated with an anti-HA antibody and protein A Sepharose beads. Immunoprecipitates were resolved on 10% SDS-polyacrylamide gels, and the resulting gel was subjected to autoradiographic detection. Densitometric analyses normalized to control cells (untreated, collected at same time) are shown in the graph, and a representative autoradiograph is shown. Drug treatment versus control: ***, p < 0.001; **, p < 0.01, one-way analysis of variance, Dunnett's multiple comparison test, n = 5 to 6.

Fig. 4.

GRK overexpression augments morphine-induced β-arrestin2-GFP translocation. HEK-293 cells were transiently transfected with HA-μOR1 (5 μg), βarr2-green fluorescent protein (GFP) (2 μg), and GRKs (2 μg) as described in detail by Groer et al. (2007). After a 30-min serum-free period, cells were treated with either DAMGO (1 μM) or morphine (10 μM). Images were taken between 10 and 15 min after treatment. Receptor expression was confirmed via immunolabeling HA-μOR on the membrane of live cells (not shown). Without coexpression of a GRK, morphine-induced translocation is barely detectable in the HEK-293 cells. When any GRK (GRK2, -3, -4, -5, or -6A) is coexpressed, morphine-induced βarr2-GFP translocation becomes readily apparent by confocal microscopy analysis (puncta formation, lower panels).