Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and β-Arrestin 2-mediated Desensitization of the μ-Opioid Receptor in Xenopus Oocytes

Celver, Jeremy; Lowe, Janet; Kovoor, Abraham; Gurevich, Vsevolod V.; Chavkin, Charles

doi:10.1074/jbc.m007437200

Cited by 90 publications

(91 citation statements)

References 28 publications

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“…ligand-bound), phosphorylated form of GPCRs. In most GPCRs, it is the carboxyl-terminal domain that undergoes ligand-dependent phosphorylation, although in the case of a number of receptors such as the m2-muscarinic receptor, phosphorylation takes place on the large third intracellular loop (27), or even the second intracellular loop, as in the case of the opioid receptor (28). In the case of the FPR, arrestin binding requires phosphorylation at multiple sites within the carboxyl-terminal domain as well as an intact DRY sequence at the beginning of the second intracellular loop, which may serve to convey the activation state of the receptor.…”

Section: Resultsmentioning

confidence: 99%

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Potter¹,

Key²,

Gurevich³

et al. 2002

Journal of Biological Chemistry

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The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/ streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to theFPRcarboxyl-terminalpeptideinaphosphorylationdependent manner, with K d values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1-382) and arrestin-3-(1-393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylationdependent manner, but with much greater affinity than the full-length arrestins, yielding K d values in the 5-50 nM range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/ V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.G protein-coupled receptors (GPCRs) 1 are the largest family of cellular surface receptors, the mediators of numerous physiological responses, and the targets of ϳ50% of therapeutic drugs (1). Their activation occurs through the binding of specific ligands to the extracellular face of the receptor, whereas cellular activation is mediated through interactions with cytoplasmic proteins, such as G proteins. Following ligand-mediated activation, GPCRs are the target of regulatory mechanisms aimed initially at terminating and then at reinitiating the signaling capacity of the cell. Furthermore, GPCR processing has also been shown to be important in the activation of Src and certain MAP kinase pathways (2). The protein arrestin was first characterized as a protein involved in GPCR desensitization following receptor phosphorylation, but has recently been demonstrated to play multiple roles in receptor function, including acting as an adapter for clathrin-mediated endocytosis and a scaffolding protein for...

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Section: Resultsmentioning

confidence: 99%

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Potter¹,

Key²,

Gurevich³

et al. 2002

Journal of Biological Chemistry

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“…4) raises the interesting possibility that ␤-arrestin could actually provide an inhibitory signal for NF-B activation and CCL2 production. A constitutively active mutant of ␤-arrestin (␤arr-R169E) has been shown to associate with phosphorylation-deficient mutants of a number of G protein-coupled receptors (43)(44)(45). To determine the role of ␤-arrestin on PAFinduced responses, transient transfectants were generated in RBL-2H3 cells coexpressing ⌬ST-PAFR with ␤arr-R169E/ green fluorescent protein conjugate (GFP-␤arr-R169E).…”

Section: Resultsmentioning

confidence: 99%

Platelet-activating Factor-induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/Calcineurin Signaling Pathways

Venkatesha

Ahamed

Nuesch

et al. 2004

Journal of Biological Chemistry

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“…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

Journal of Biological Chemistry

115

112

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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...

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Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and β-Arrestin 2-mediated Desensitization of the μ-Opioid Receptor in Xenopus Oocytes

Cited by 90 publications

References 28 publications

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Platelet-activating Factor-induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/Calcineurin Signaling Pathways

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

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