Regulation of μ-opioid receptors: desensitization, phosphorylation, internalization, and tolerance

Abstract

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

Fig. 1.

General scheme of MOR regulation following binding of an efficacious agonist such as [Met]⁵enkephalin. The time scales for each process are shown (log scale). Phosphorylation by G protein receptor kinase (GRK) is very rapid, saturating in less than 20 seconds. Arrestin binding saturates in several minutes, and desensitization reaches steady state in approximately 5 minutes. The steady state of rapid desensitization represents the equilibrium between the forward desensitizing process, presumably phosphorylation and arrestin binding (other kinases may be involved, see Section V.D–V.G) and dephosphorylation at the cell surface (see Sections I, V, and VI). Endocytosis reaches steady state in approximately 30 minutes and recycling over approximately 60 minutes, although this varies for different splice variants. The present review defines desensitization as the rapid process preceding significant endocytosis (approximately 2–5 minutes); short-term tolerance includes endocytosis and other mechanisms (up to 1 day); and long-term tolerance (greater than 1 day) presumably involves multiple regulatory processes.

Fig. 2.

Agonist binding to MORs can result in the activation of multiple downstream pathways. Different agonists can selectively activate one or a number of these pathways that give rise to agonist-selective signaling through a single receptor subtype. G protein-dependent processes include the regulation of ion channels and inhibition of adenylyl cylase. Mechanisms involved in desensitization may involve selective activation of one or another kinase dependent pathway. G protein-independent processes, including the steps leading to endocytosis and interactions with scaffolding molecules and kinases, may influence MOR signaling by both direct and potentially indirect mechanisms.

Fig. 3.

Ligand bias at MOR. The intrinsic efficacies (operational model) of a range of structurally dissimilar MOR agonists to activate [³⁵S]GTPγS binding and arrestin recruitment was determined and the bias factor (β) calculated according to the method of Rajagopal et al. (2011). Reproduced from Rivero et al. (2012).

Fig. 4.

Summary of putative (shaded) and established agonist-induced phosphorylation sites on the MOR and associated kinases. There is moderate to strong evidence for the colored residues as follows: Tyr106, Tyr166, probably phosphorylated by tyrosine kinase (McLaughlin and Chavkin, 2001); Thr180, probably phosphorylated by GRK3 (Celver et al., 2001); Ser266, probably phosphorylated by CaMKII (Koch et al., 1997, 2000); Tyr336, phosphorylated by Src kinase (Zhang et al., 2009); Ser355, Thr357, one or both phosphorylated (Wang et al., 2002; Lau et al., 2011) by GRK2 (Wang 2000); Ser363, constitutive phosphorylation (Doll et al., 2011) by PKC (Feng et al., 2011); Thr370, Ser375, phosphorylation directly shown (Schulz et al., 2004; Doll et al., 2011); Thr376, Thr379, one or both phosphorylated (Lau et al., 2011); Thr383, Thr394, phosphoylation was predicted but not directly shown (Pak et al., 1997), and not observed (El Kouhen et al., 2001; Lau et al., 2011).

Fig. 5.

Summary of MOR phosphorylation and enzyme interactions leading to desensitization and endocytosis. (A) For agonists with high relative efficacy for endocytosis, G protein dissociation and conformational changes favorable to GRK phosphorylation drive desensitization and endocytosis. GRK2 and GRK3 appear to be the major isoforms involved (see Desensitization and Tolerance Are Both Associated with Reduction of Functional Receptors). Arrestin binding requires GRK phosphorylation, and both β-arrestin2 and β-arrestin1 can interact with MOR to promote endocytosis (see Biased Agonism and μ-Opioid Receptor Regulation; Groer et al., 2011). It is not certain if phosphorylation events, β-arrestin binding, or both produce uncoupling of MOR signaling (desensitization), but the time course appears to follow arrestin binding more closely. Strong internalizing agonists activate phospholipase D2 (Section V.G), but it is not certain whether this is required for endocytosis (Arttamangkul et al., 2012). There is some evidence that ERK1/2-dependent mechanisms may desensitize MOR by both arrestin-independent (Gβγ) and arrestin-dependent mechanisms. Other kinases may also be important, including CAMKII and PKC. GRK phosphorylation is rapidly reversible at the cell surface, but the rate of reversal at other potential phosphorylation sites or their requirements for endocytosis is unknown (see Phosphorylation and μ-Opioid Receptor Regulation). (B) Agonists with relatively low efficacy for endocytosis weakly and slowly phosphorylate GRK substrates on MOR and induce weak association with β-arrestin2 (see Desensitization and Tolerance Are Both Associated with Reduction of Functional Receptors and Biased Agonism and μ-Opioid Receptor Regulation). However, there is good evidence that PKC-dependent mechanisms, possibly via direct phosphorylation of MOR at Thr370 (Doll et al., 2011), contribute to desensitization by agonists such as morphine (Section V.D). PKC also appears to recruit JNK-dependent desensitization mechanisms for agonists such as morphine but not for high endocytosis efficacy agonists (Melief et al., 2010). It is tempting to speculate for agonists with low efficacy for endocytosis that PKC and other kinases can readily interact with the intracellular domain of MOR when it is not occluded either by G proteins or arrestins. Whether these events are rapidly reversible (as is GRK dephosphorylation) is not known.

Fig. 6.

Summary of adaptations that might contribute to MOR tolerance after chronic exposure to morphine (but perhaps not other agonists). (A) Functional analyses indicate >80% loss of functional MOR is required to account for tolerance after chronic morphine, but this is not accounted for by loss of total MOR binding (see Primary Structure and Structural Diversity of μ-Opioid Receptors). (B) An enhanced rate of MOR desensitization coupled with impaired resensitization should shift the equilibrium toward increased desensitized MOR (Section VII). The dependence of impaired MOR resensitization on GRK and arrestin could explain the loss of morphine tolerance observed β-arrestin2 knockouts (Section VII.B). (C) Although increased endocytosis could contribute to loss of functional MOR, there is evidence (Quillinan et al., 2011) that does not support this idea (Section VII.A and VII.B). (D) Blocking PKC activity reverses tolerance in vivo and in vitro (Section V.G) raising the possibility that persistent PKC phosphorylation of MOR may be required for loss of MOR function. (E) The dependence of impaired MOR resensitization on GRK and arrestin suggests that enhanced interactions may contribute to persistent desensitization. However, there is little direct evidence to support this possibility (Section VII.A, VII.E, VII.F). (F) Reduced recycling of MOR has been observed in locus coeruleus neurons and this should produce accumulation of intracellular MOR. While this occurs in some neurons, it has not been observed in others (see Primary Structure and Structural Diversity of μ-Opioid Receptors).