Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes

Drakopoulos, Antonios; Köszegi, Zsombor; Lanoiselée, Yann; Hübner, Harald; Gmeiner, Peter; Calebiro, Davide; Decker, Michael

doi:10.1021/acs.jmedchem.9b02011

Cited by 17 publications

(18 citation statements)

References 104 publications

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“…Indeed, the group of Decker showed that by tagging GNTI at the 5’‐position with sulfo‐Cy3 and sulfo‐Cy5 fluorophores, κOR selective fluorescent ligands 94a‐b were obtained (Scheme 53A). However, in this study, they could not detect any dimerization of κORs and the authors suggest that the fluorescent ligands are mostly present as freely diffusing monomers on the surface of transiently transfected CHO cells [193] …”

Section: Fluorescent Ligands For Gpcrscontrasting

confidence: 58%

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Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Borgarelli

Klingl

Escamilla-Ayala

et al. 2021

Chemistry A European J

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Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in‐depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target‐tailored small‐molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high‐precision optical techniques to image with an unprecedented resolution at a single‐molecule level, helped unraveling many of the conundrums related to plasma proteins’ life‐cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage‐gated ion channels, ligand‐gated ion channels and G‐coupled protein receptors.

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Section: Fluorescent Ligands For Gpcrscontrasting

confidence: 58%

“…However, in this study, they could not detect any dimerization of kORs and the authors suggest that the fluorescent ligands are mostly present as freely diffusing monomers on the surface of transiently transfected CHO cells. [193] Scheme 52. Opioid receptor ligands and fluorescent ligands.…”

Section: Fluorescent Ligands For Melatonin Receptors (Mtrs)mentioning

confidence: 99%

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Borgarelli

Klingl

Escamilla-Ayala

et al. 2021

Chemistry A European J

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“…However, for κOR, the yellow fraction increases from a low value of 10% at a density of 14/µm 2 to a high value of 46% for densities of > 100/µm 2 , supporting the expected density dependence of the κOR dimer/cluster fraction. As supported by previous studies on GPCRs and ORs, we assume in the following that the κOR is either monomeric or dimeric [2][3][4][5][6][7][11][12][13][14].…”

Section: Dissociation Constant Of κOrmentioning

confidence: 99%

“…µOR seems to be monomeric when unliganded and switches to a dimeric state when the ligand DAMGO is bound, but interestingly stays monomeric upon binding of morphine [ 11 , 12 ]. κOR has been observed to be primarily monomeric [ 13 ]. However, for these studies, experiments were performed at low densities < 1/µm 2 .…”

Section: Introductionmentioning

confidence: 99%

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Cechova

Macik

Barthès

et al. 2021

Cell. Mol. Life Sci.

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Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

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“…μOR seems to be monomeric when unliganded, but switches to a dimeric state when the ligand DAMGO is bound, but interestingly stays monomeric upon binding of morphine [11, 12]. κOR has been observed to be primarily monomeric [13]. However, for these studies, experiments were performed at low densities < 1 / µm 2 .…”

Section: Introductionmentioning

confidence: 99%

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Cechova

Barthès

Jung

et al. 2020

Preprint

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Opioid receptors (ORs) have been observed as homo-and heterodimers, but it is unclear if the dimers are stable under physiological conditions. Here we use three live-cell imaging approaches to assess dimerization of ORs at different expression levels. At high membrane densities, a split GFP assay reveals that OR dimerizes, while OR and OR stay monomeric. In contrast, singlemolecule imaging showed no OR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities 100x higher than conventional single-molecule imaging. We observe that OR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, OR and OR stay monomeric even at the highest densities covered. The observation of long-lasting OR dimers but not higher order aggregates suggests that OR dimerization is a specific effect and not a result of increasing expression. IntroductionORs are G protein-coupled receptors (GPCRs) from class A with three genes coding for the OR, OR, and OR. Based on pharmacological profiles, more subtypes were proposed, which can be explained by the existence of splicing variants, posttranslational modifications and/or direct interactions between receptors. Dimerization of ORs has been covered in multiple studies, yet the conclusions were contradictory, as for many other GPCRs, likely due to the use of differing methodological approaches and experimental conditions [1].So far, the major techniques to assess dimerization of ORs were co-immunoprecipitation followed by Western blotting, and bioluminescence resonance energy transfer (BRET) [2][3][4][5][6]. Both are bulk techniques, where the signal is obtained from a large population of cells. Not only is the major part of the signal caused by a small, highly expressing fraction of the cells, but in addition, these few cells express the receptors at the highest density. Therefore, the signal mainly reflects the receptor's behavior at a membrane density that is far above the physiological range, but cannot capture the state of the receptors at low membrane densities, as they prevail in many cells in vivo.In this work, we quantitatively measured OR dimerization using different microscopy approaches that work at different densities. Our starting point was a split GFP fluorescence complementation assay in cells with expression levels of 50-100 / µm 2 . This assay gave us a first indication that the OR has a tendency to dimerize, while the OR and OR resembled the monomeric control, which was the PDGFR transmembrane domain (PDGFRTM). However, with conventional dualcolor single-molecule imaging at densities below 5 / µm 2 , we did not observe a significantly higher dimerization for OR than for the other ORs. Therefore, we turned to a recently developed technique called PhotoGate that allows tracking of single molecules in an originally crowded environment by controlling the density of fluorescent molecules in a re...

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Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes

Cited by 17 publications

References 104 publications

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

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