Abstract

Morphine and other opiates mediate their effects through activation of the μ-opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, β-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of β-arrestin, β-arrestin1 and β-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting β-arrestin2 specifically. In this study, we examine the different contributions of β-arrestin1- and β-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both β-arrestins. Here we show that morphine only recruits β-arrestin2, whereas the MOR-selective enkephalin [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), recruits either β-arrestin. We show that β-arrestins are required for receptor internalization and that only β-arrestin2 can rescue morphine-induced MOR internalization, whereas either β-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, β-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of β-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and β-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.

Highlights

  • Tein-coupled receptor (GPCR), the mediated largely through activation of ␮-opioid receptors (MORs) is subject to regulation paradigms that include phosphorylation by GPCR kinases (GRKs) and subsequent interactions with ␤-arrestins (␤-arrestin1, known as arrestin2, and ␤-arrestin2, known as arrestin3). ␤-Arrestins can initiate receptor internalization, which in turn can promote both receptor down-regulation and resensitization [1,2,3]. ␤-Arrestins can facilitate these regulatory events by scaffolding ubiquitination machinery, such as E3 ligases, to GPCRs, as has been shown for the ␤2 adrenergic receptor (␤2AR), V2 vasopressin receptor, and the chemokine receptor (CXCR4) (4 – 6), this has not been demonstrated for the MOR

  • We show for the first time quantitative evidence that morphine, even in the presence of excess GRK2, exclusively leads to MOR recruitment of ␤-arrestin2 and not ␤-arrestin1, whereas DAMGO promotes recruitment of both ␤-arrestins (Fig. 2)

  • We show that ␤-arrestins are required for MOR internalization (Fig. 3) and that either ␤-arrestin isoform can facilitate this event if the agonist is capable of promoting receptor interactions with that particular ␤-arrestin (Fig. 4)

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Summary

EXPERIMENTAL PROCEDURES

DAMGO (Tocris, Ellisville, MO) and morphine (morphine sulfate pentahydrate; Sigma) were dissolved in distilled water to 10 mM. 10 mM stock solutions were diluted in PBS to working concentrations. Rat ␤-arrestin (gi949985) and ␤-arrestin (gi949986) were tagged on the C terminus with enhanced green fluorescent protein (␤arr2-GFP and ␤arr1GFP) for microscopy studies or with GFP2 for BRET studies (␤arr1-GFP2 and ␤arr2-GFP2). Rat ␤-arrestin and ␤-arrestin were Myc-tagged on the N terminus (Myc-␤arr and Myc␤arr2) for internalization rescue studies. MEFs were transduced with HA-MOR using murine stem cell virus. A FACS Aria flow cytometer was used to select for high expressing cells (top ϳ25%) using an anti-HA AlexaFluor 488 conjugate antibody (1:200; Invitrogen). Uniform cell surface expression of the HA-MOR under basal conditions was confirmed by confocal microscopy (Fig. 2)

Radioligand Binding
MOR Internalization
MOR Ubiquitination
MOR Dephosphorylation
Graphical Modifications to Images
RESULTS
These effects were quantitated using the cell surface biotinylation
DISCUSSION
Full Text
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