Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

Morse, Megan; Tran, Elizabeth; Sun, Haiyan; Levenson, Robert; Ye, Fang

doi:10.1371/journal.pone.0025643

Cited by 44 publications

(46 citation statements)

References 59 publications

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“…Morse et al (2011) examined a range of endogenous and exogenous opioids in HEK293 cells using dynamic mass redistribution to measure MOP global responses. In that study, all endogenous opioids, except dynA 1-13, exhibited similar profiles.…”

Section: Discussionmentioning

confidence: 99%

Biased Agonism of Endogenous Opioid Peptides at theμ-Opioid Receptor

et al. 2015

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Biased agonism is having a major impact on modern drug discovery, and describes the ability of distinct G protein-coupled receptor (GPCR) ligands to activate different cell signaling pathways, and to result in different physiologic outcomes. To date, most studies of biased agonism have focused on synthetic molecules targeting various GPCRs; however, many of these receptors have multiple endogenous ligands, suggesting that "natural" bias may be an unappreciated feature of these GPCRs. The m-opioid receptor (MOP) is activated by numerous endogenous opioid peptides, remains an attractive therapeutic target for the treatment of pain, and exhibits biased agonism in response to synthetic opiates. The aim of this study was to rigorously assess the potential for biased agonism in the actions of endogenous opioids at the MOP in a common cellular background, and compare these to the effects of the agonist D-Ala2-N-MePhe4-Gly-ol enkephalin (DAMGO). We investigated activation of G proteins, inhibition of cAMP production, extracellular signal-regulated kinase 1 and 2 phosphorylation, b-arrestin 1/2 recruitment, and MOP trafficking, and applied a novel analytical method to quantify biased agonism. Although many endogenous opioids displayed signaling profiles similar to that of DAMGO, a-neoendorphin, Met-enkephalin-Arg-Phe, and the putatively endogenous peptide endomorphin-1 displayed particularly distinct bias profiles. These may represent examples of natural bias if it can be shown that they have different signaling properties and physiologic effects in vivo compared with other endogenous opioids. Understanding how endogenous opioids control physiologic processes through biased agonism can reveal vital information required to enable the design of biased opioids with improved pharmacological profiles and treat diseases involving dysfunction of the endogenous opioid system.

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Section: Discussionmentioning

confidence: 99%

Biased Agonism of Endogenous Opioid Peptides at theμ-Opioid Receptor

et al. 2015

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“…Our results revealed that the inhibition of ERK1/ ERK2 with U0126, a potent MEK inhibitor, significantly reduced the maximal amplitude of the SPR responses induced by both MOP agonists. Other groups previously showed that the maximal responses triggered by morphine and DAMGO were barely affected by U0126 (Codd et al, 2011;Morse et al, 2011). Although Epic (Morse et al, 2011) and SPR (current study) are dynamic mass redistribution optical biosensors, their detecting systems are slightly different.…”

Section: Discussionmentioning

confidence: 55%

“…Human embryonic kidney (HEK)-293 cells stably expressing Flagtagged human MOP (HEK FlagMOP), with a receptor density of 2.5 pmol/mg cell proteins (Morse et al, 2011), were kindly provided by Dr. Mark von Zastrow (University of California, San Francisco, CA). The cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 50 mg/l gentamicin in a humidified atmosphere of 95% air and 5% CO 2 .…”

Section: Cell Culturementioning

confidence: 99%

“…Recent reports demonstrated the usefulness of whole-cell biosensors to study functional selectivity of several GPCRs. For example, Stallaert et al (2012) used an impedance-based system to assess functional selectivity at the b 2 adrenergic receptor, whereas Morse et al (2011Morse et al ( , 2013 used an optical-based system to assess opioid receptor signaling. We previously showed that SPR represents a valuable label-free technique to monitor GPCR signaling and apoptosis (Cuerrier et al, 2008;Chabot et al, 2009;Maltais et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Label-Free Monitoring ofμ-Opioid Receptor–Mediated Signaling

et al. 2014

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In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the m-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-DAla-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Ga i , cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine-and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 ), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-CysTyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 ) induces similar conformational changes as naltrexone at the Ga i -bg interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of labelfree methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable.

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“…To visualize the patterns of DMR signals arising from GPCR agonists under different conditions, we used our recently developed similarity analysis approach. [22][23][24] This approach converts a ligand-induced DMR into a multidimensional coordinate such that all ligands tested can be compared using similarity analysis. Similarity analysis is a powerful means to determine the relationships (i.e., similarity or distances) among different biological responses, particularly for large sets of biological data.…”

Section: Dmr Profiling Of Endogenous Gpcrsmentioning

confidence: 99%

Dynamic Mass Redistribution Assay Decodes Differentiation of a Neural Progenitor Stem Cell

et al. 2012

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Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-proteincoupled receptor agonist library revealed that a subset of receptors, including dopamine D 1 and D 4 receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.

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Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

Cited by 44 publications

References 59 publications

Biased Agonism of Endogenous Opioid Peptides at theμ-Opioid Receptor

Biased Agonism of Endogenous Opioid Peptides at theμ-Opioid Receptor

Label-Free Monitoring ofμ-Opioid Receptor–Mediated Signaling

Dynamic Mass Redistribution Assay Decodes Differentiation of a Neural Progenitor Stem Cell

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