Phosphorylation and Agonist‐Specific Intracellular Trafficking of an Epitope‐Tagged μ‐Opioid Receptor Expressed in HEK 293 Cells

Arden, James; Segredo, Veronica; Wang, Zaijie; Lameh, Jelveh; Sadée, Wolfgang

doi:10.1046/j.1471-4159.1995.65041636.x

Cited by 276 publications

(156 citation statements)

References 23 publications

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“…In particular, our results suggest that the distal portion of the carboxyl-terminal cytoplasmic domain of DOR is not required for regulated endocytosis but functions as an endocytic brake that is "released" by phosphorylation in order to allow endocytosis of full-length receptors to occur. At first glance, this hypothesis is reminiscent of a previous study suggesting that the cytoplasmic tail of the -opioid receptor inhibits endocytosis (29). However, in this case, truncation of the corresponding tail domain caused constitutive endocytosis of receptors (i.e.…”

Section: Discussionmentioning

confidence: 73%

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

Whistler¹,

Tsao²,

Zastrow³

2001

Journal of Biological Chemistry

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The ␦-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor downregulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxylterminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent proteintagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptormediated recruitment of ␤-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylationdefective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.

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Section: Discussionmentioning

confidence: 73%

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

Whistler¹,

Tsao²,

Zastrow³

2001

Journal of Biological Chemistry

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“…In HEK293 cells, phosphorylation of the p-opioid receptor was increased only 80% above control (Arden et al, 1995). Furthermore, the hippocampal slice has a heterogeneous population of neurons, not all of which necessarily have averages of the control values for that experiment.…”

Section: Figmentioning

confidence: 95%

Agonist‐Induced Phosphorylation of the κ‐Opioid Receptor

Appleyard

Patterson

Jin³

et al. 1997

Journal of Neurochemistry

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Antipeptide antibodies against the K-opioid receptor were used to test whether acute or chronic exposure to K agonists altered the phosphorylation state of the K-Opioid receptor. Immunoprecipitation of the K receptor from guinea pig hippocampal slices preincubated in [ 32P]orthophosphoric acid revealed a basal phosphorylation of the K-opioid receptor. The amount of 32P incorporation into the receptor was increased following a 75-mm treatment with the K agonist U50,488H. This effect was blocked by the selective K receptor antagonist norbinaltorphimine. The time course of this change in the phosphorylation state of the receptor correlated with a desensitization of the electrophysiological response to K agofists measured in the dentate gyrus of hippocampal slices. The phosphorylation state of the K-Opioid receptor was also elevated in brain slices from guinea pigs made tolerant to U50,488H by 5 days of continuous exposure and then maintained in K agonist to avoid acute opiate withdrawal. The results of this study show that the Kopioid receptor was phosphorylated in an agonist-dependent manner in brain slices taken from untreated and U50,488H-tolerant animals.

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“…17 in the case of the µ-opioid receptor, we and others have reported that compounds can display clearly different profiles in G-protein activation and β-arrestin recruitment, with morphine exhibiting low emax values in β-arrestin recruitment/receptor internalization but high emax values in assays measuring G-protein activation. [18][19][20][21] However, emax values are not always a good indicator of efficacy. Herein, we have shown that some ligands do show agonist-directed conformational states; for instance, morphine does not differentiate from the other ligands in terms of efficacy (tau) for G-protein activation but does differentiate in terms of efficacy (emax) in the β-arrestin assay.…”

Section: Drugs and Chemicals Usedmentioning

confidence: 99%

Understanding the Effect of Different Assay Formats on Agonist Parameters: A Study Using the µ-Opioid Receptor

et al. 2011

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IntroductIonG -protein-coupled receptors (Gpcrs) are the largest superfamily of cell surface signal transduction molecules. they are important targets in drug discovery, with an estimated 25% of prescription drugs exerting their effects through them. 1 Gpcrs exert many of their effects by activation of heterotrimeric G proteins (Gi, Gs, Gq). Following activation, G proteins divide into respective α and βγ subunits, both of which have the ability to activate or inhibit effector molecules, such as adenylate cyclase and phospholipase c. the receptor then becomes phosphorylated on intracellular serine and threonine residues by Gpcr kinases (GrKs). 2,3 the phosphorylated receptor is then bound by β-arrestin, which sterically prevents further G-protein interaction and recruits other proteins such as clathrin, which are involved in receptor internalization. in addition, β-arrestin has been shown to function as a signaling intermediate and, for instance, is involved in the activation of the MApK pathway. 4,5 Historically, within the drug discovery environment, Gpcr agonists have been discovered through traditional second messenger assay formats, which measure the activation of G proteins (e.g., [ 35 s]Gtpγs binding) and subsequent signaling events (e.g., inhibition of forskolin-stimulated cAMp production). However, in recent years, pressure to develop more robust and high-throughput assays, with higher Z′ values and the discovery of non-G-protein-mediated signaling through Gpcrs, has led to the development of alternative assay formats, such as β-arrestin recruitment. in particular, the discoverx pathHunter assay technology used in this report uses an enzyme complementation system to detect the interaction between a tagged Gpcr and tagged β-arrestin. 6,7 Although this assay is being used with increasing frequency, the pharmacologic values generated have not been thoroughly investigated and put into context with traditional pharmacological models.Agonists have two properties: affinity (K A ), which is a purely drug-dependent parameter and determines how well the agonist binds to the receptor (and therefore should be equivalent for all functional endpoints), and efficacy (tau), which measures the efficiency of the transduction of binding into a response and is dependent on the ligand and the tissue/signaling pathway involved (and therefore is likely to differ for different functional endpoints). the translation of occupancy into effect is what is measured in the form of an agonist-concentration/effect ([A]/e) curve. the ability to discriminate between agonists with different efficacies is of critical importance in drug discovery. However, overexpressing recombinant systems can lead to the overestimation of the potency and efficacy of compounds, and in particular, 1 discovery Biology, pfizer inc., Kent, uK. the correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. often, new assay technologies are assimilated into the drug discovery environment without full consideration of t...

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Phosphorylation and Agonist‐Specific Intracellular Trafficking of an Epitope‐Tagged μ‐Opioid Receptor Expressed in HEK 293 Cells

Cited by 276 publications

References 23 publications

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

Agonist‐Induced Phosphorylation of the κ‐Opioid Receptor

Understanding the Effect of Different Assay Formats on Agonist Parameters: A Study Using the µ-Opioid Receptor

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