Role for the C-Terminus in Agonist-Induced μ Opioid Receptor Phosphorylation and Desensitization

117

126

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between -opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though -receptors and ␦-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.Opioid alkaloids, as well as endogenous opioid peptides, exert their multiple biological effects on target tissues by interacting with specific cell surface receptors including the ␦-, -, and -opioid receptors (1). These opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs).1 The -opioid receptor (MOR) serves as the principle physiological target for most clinically important opioid analgesics, such as morphine and fentanyl (2, 3). Although many opioid alkaloids exert their pharmacological effects via MOR, their binding affinity for the receptor and potency to activate the receptor do not always correspond to their abilities to induce tolerance (4 -8). This suggests that other cellular processes that modulate MOR responsiveness, such as receptor desensitization and internalization, may contribute to opioid tolerance and dependence. Like many other GPCRs, opioid receptors are regulated by agonist-dependent processes and undergo receptor phosphorylation, desensitization, internalization, and down-regulation (1). Interestingly, in addition to the subtype-specific regulation of opioid receptors (9 -12), individual opioid receptors are differentially regulated by distinct opioid agonists (5-8, 13, 14). In the case of MOR, opioid agonists demonstrating equivalent ability to activate receptor signaling exhibit remarkable differences in their abilities to functionally desensitize (5, 6) and induce internalization of the receptor in both transfected cells and neurons (7,8,(13)(14)(15). However, the detailed molecular events underlying this differential regulation of MOR by distinct agonists remain unclear. Using chimeric, truncated, or mutated opioid receptors, several studies reported the crucial role of the C-tail of opioid receptors in regulating their activities and trafficking (11, 16 -20). Whereas several potential phosphorylation sites were suggested to be involved in regulating the activity and trafficking of opioid receptors, the actual ...

Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Ser363, Thr370, and Ser375 Residues within the Carboxyl Tail Differentially Regulates μ-Opioid Receptor Internalization

Kouhen

Burd

Erickson-Herbrandson

et al. 2001

117

126

“…Two research groups (14,21) . This discrepancy of identified phosphorylation sites in different cell lines may indicate that the agonist-induced phosphorylation sites are celltype specific.…”

Section: Resultsmentioning

confidence: 99%

“…The time course and dose-response relationships between phosphorylation and agonist-induced desensitization display parallels in CHO cells stably expressing MOR (13). The absence of agonist-induced phosphorylation by the mutation of putative phosphorylation sites to alanine completely blocks or significantly attenuates the agonist-induced receptor desensitization (14,15) and internalization (16). The enhanced phosphorylation of MOR has been correlated with the desensitization of MOR in thalamus of rats chronically treated with morphine (17).…”

mentioning

confidence: 99%

μ-Opioid Receptor Desensitization

Qiu

Law

Loh

2003

It is generally accepted that the internalization and desensitization of -opioid receptor (MOR) involves receptor phosphorylation and ␤-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser 363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of ␤-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent ␤-arrestin recruitment play important roles in the resensitization of internalized receptors.The regulation of G protein-coupled receptors (GPCRs) 1 signaling highlights the complex relationship between multiple mechanisms acting at different levels of signal propagation. Following the agonist activation of the receptor, G proteincoupled receptor kinases (GRKs) are recruited to the membrane and phosphorylate the agonist-activated GPCRs. GRKmediated receptor phosphorylation enhances the interaction between GPCRs and cytoplasmic proteins, arrestins, which uncouple the GPCRs from G proteins and also target the GPCRs to clathrin-coated pits for internalization (endocytosis). The internalized receptors can subsequently be dephosphorylated and recycled back to the plasma membrane, which contributes to resensitization. Alternatively, the internalized receptors can be targeted to lysosome for degradation (1-4). Receptor desensitization, the progressive loss of receptor function under continued exposure to an agonist, occurs in many GPCRs. Desensitization can be a consequence of multiple processes of receptor uncoupling, internalization, degradation, and recycling. Therefore, phosphorylation of agonist-activated receptors and subsequent arrestin recruitment are important processes in the modulation of GPCRs responsiveness.However, multiple studies challenge the critical role phosphorylation played in desensitization and internalization of many GPCRs. Desensitization and internalization of human parathyroid hormone receptor (5), AT 1A angiotensin receptor (6), and metabotropic gluta...

“…This suggests that the differences in MOR dephosphorylation rate are due to the agonist's ability to recruit ␤-arrestin1 to the receptor. Importantly, the possible continued phosphorylation of other residues cannot be discounted, including threonines 180 and 394, because both of these residues have been shown to be important for DAMGO-induced MOR desensitization (24,25). It is possible that the collective pattern of MOR phosphorylation will be critical in the overall functional state of the MOR.…”

Section: Discussionmentioning

confidence: 99%

Agonist-directed Interactions with Specific β-Arrestins Determine μ-Opioid Receptor Trafficking, Ubiquitination, and Dephosphorylation

Groer

Schmid

Jaeger

et al. 2011

115

112

Morphine and other opiates mediate their effects through activation of the -opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, ␤-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of ␤-arrestin, ␤-arrestin1 and ␤-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting ␤-arrestin2 specifically. In this study, we examine the different contributions of ␤-arrestin1-and ␤-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both ␤-arrestins. Here we show that morphine only recruits ␤-arrestin2, whereas the MOR-selective enkephalin [D-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]enkephalin (DAMGO), recruits either ␤-arrestin. We show that ␤-arrestins are required for receptor internalization and that only ␤-arrestin2 can rescue morphine-induced MOR internalization, whereas either ␤-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, ␤-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of ␤-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and ␤-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.Morphine and other opiates are among the most clinically useful analgesics, and their actions are mediated largely through activation of -opioid receptors (MORs).3 As a G protein-coupled receptor (GPCR), the MOR is subject to regulation paradigms that include phosphorylation by GPCR kinases (GRKs) and subsequent interactions with ␤-arrestins (␤-arrestin1, also known as arrestin2, and ␤-arrestin2, also known as arrestin3). ␤-Arrestins can then initiate receptor internalization, which in turn can promote both receptor down-regulation and resensitization (1-3). ␤-Arrestins can also facilitate these regulatory events by scaffolding ubiquitination machinery, such as E3 ligases, to GPCRs, as has been shown for the ␤ 2 adrenergic receptor (␤ 2 AR), V2 vasopressin receptor, and the chemokine receptor (CXCR4) (4 -6), however this has not been demonstrated for the MOR. MOR regulation has been shown to be contingent upon the particular agonist acting at the receptor as morphine promotes different regulatory events than other opioid ligands, including the D-enkephalin analog, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO), fentanyl, methadone, and etorphine, although all of these ligands are full agonists at the MOR with respect to G protein coupling (7). The difference between agonists was first recognized when Arden et al. (8) observed that although DAMGO promotes robust internalizatio...