Changes of the Membrane Lipid Organization Characterized by Means of a New Cholesterol-Pyrene Probe (original) (raw)
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-Opioid receptor activation in live cells
The FASEB Journal, 2008
Interaction of the -opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80؎10%) were characterized by a diffusion coefficient D MOP, 1 ؍ (4؎2) ؋ 10 ؊11 m 2 s ؊1 , compared with the slowly moving fraction, D MOP, 2 ؍ (4؎2) ؋ 10 ؊12 m 2 s ؊1 . On stimulation with selected agonists ([D-Ala 2 ,N-MePhe 4 ,Gly-ol 5 ]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.-Vukojević , V., Ming, Y., D'Addario, C., Hansen, M., Langel, Ü ., Schulz, R., Johansson, B., Rigler, R., Terenius, L. -Opioid receptor activation in live cells. FASEB J. 22, 3537-3548 (2008)
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008
Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR⁎) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR⁎ is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR⁎ was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.
Methods in molecular biology (Clifton, N.J.), 2015
The development of new and improved opioid analgesics requires high-throughput screening (HTS) methods to identify potential therapeutics from large libraries of lead compounds. Here we describe two simple, real-time fluorescence-based assays of μ-opioid receptor activation that may be scaled up for HTS. In AtT-20 cells expressing the μ-opioid receptor (MOPr), opioids activate endogenous G protein gated inwardly rectifying K channels (GIRK channels), leading to membrane hyperpolarization. In Chinese hamster ovary cells expressing MOPr, adenylyl cyclase activation via forskolin results in membrane hyperpolarization, which is inhibited by opioids. Changes in membrane potential can be measured using a proprietary membrane potential-sensitive dye. In contrast to many HTS methods currently available, these assays reflect naturalistic coupling of the receptor to effector molecules.
Localization of the κ Opioid Receptor in Lipid Rafts
Journal of Pharmacology and Experimental Therapeutics, 2006
Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined whether opioid receptors (KOR) in human placenta and FLAG (DYKDDDDK)-tagged human KOR (FLAG-hKOR) expressed in Chinese hamster ovary (CHO) cells are localized in lipid rafts and whether changes in cholesterol contents affect hKOR properties and signaling. Lipid rafts were prepared from placenta membranes and CHO cells expressing FLAG-hKOR using the Na 2 CO 3 method and fractionation through a sucrose density gradient. The majority of the KOR in the placenta and FLAG-hKOR in CHO cells, determined by [ 3 H]diprenorphine binding and/or immunoblotting with an anti-FLAG antibody, was present in low-density fractions, coinciding with high levels of caveolin-1 and cholesterol, markers of lipid rafts, which indicated that the KOR is localized in lipid rafts. Pretreatment with 2% methyl -cyclodextrin (MCD) reduced cholesterol content by ϳ48% and changed the cells from spindle-shaped to spherical. MCD treatment disrupted lipid rafts, shifted caveolin-1 and FLAG-hKOR to higher density fractions, increased the affinity of (Ϫ)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488H) for the hKOR, and greatly increased U50,488H-induced [ 35 S]guanosine 5Ј-O-(3-thio)triphosphate binding and p42/44 mitogen-activated protein kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with G␣ i proteins and MCD treatment reduced caveolin-1 associated with G␣ i proteins, which may contribute to the enhanced agonist-induced G protein activation. Caveolin-1 also immunoprecipitated with FLAG-hKOR, but MCD treatment had no effect on the association. Thus, the KOR is located in lipid rafts and its localization in the microdomains greatly affects coupling to G proteins. Plasma membranes were traditionally viewed as uniform lipid bilayers and G protein-coupled receptors (GPCRs), G proteins, and membrane-bound effectors were randomly distributed in plasma membranes. In recent years, the concept that lipid rafts function as microdomains in plasma membranes to concentrate signaling molecules for regulated activation by related receptors has gained increasing acceptance (Pike, 2003; Chini and Parenti, 2004; Cohen et al., 2004). Lipid rafts are microdomains of cell membranes highly enriched in cholesterol and sphingolipids in the outer layer. Brown and Rose (1992) proposed the following operational definition of lipid rafts: when cell membranes were solubi-lized with ice-cold nonionic detergents such as Triton X-100 (1% for 1 h at 4°C) followed by sucrose density gradient centrifugation; lipid rafts are resistant to detergent solubilization and float in the lighter fractions, whereas the bulk of the solubilized cellular lipids and proteins are in the high-density fractions (Brown and Rose, 1992). There are two types of lipid rafts: planar lipid rafts and caveolae. Caveolae, a specialized subtype of lipid rafts, are flask-shaped invaginations in the plasma membrane and are enriched in caveolins, 21-to 24-kDa integral membrane proteins (Razani et al., 2002). Many signaling molecules including some GPCRs, G␣ proteins, growth factor receptors, protein kinase C, and adenylyl cyclase were found to be enriched in or recruited into lipid rafts (Pike, 2003; Chini and Parenti, 2004). Growing evidence has indicated that lipid rafts are crucial in the regulation of GPCRs in signal transduction and in exocytic and endocytic pathways (Pike, 2003; Chini and Parenti, 2004). Cholesterol has been shown to play a critical role in assem
A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells
2012
Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.
A Continuous, Fluorescence-based Assay of -Opioid Receptor Activation in AtT-20 Cells
Journal of Biomolecular Screening, 2013
Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.
Journal of supramolecular structure, 1979
Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the alpha-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra of fluorescence lifetimes, are highly sensiti...
Affinity labelling of frog brain opioid receptors by dynorphin (1–10) chloromethyl ketone
Neuropeptides, 1997
It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphinil_10/-Glyll chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the ~:-opioid binding sites labelled by [SH]ethylketocyclazocine (Ki = 200 nM), whereas its binding to the ([3H]dihydromorphine) and to the ~ sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 ~M DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The K-preferring binding properties of the compound suggest that it could serve as an affinity label for the K-type of opioid receptors.
Journal of Membrane Biology, 2008
The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein-coupled receptor adopts a characteristic a-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the a-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP-HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.
Oligomerization of opioid receptors
Methods, 2002
Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory Gproteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K þ channels and the inhibition of Ca 2þ channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions. Ó