title: Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol.
abstract: 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.
authors.affiliation: IPBS, MR5089 CNRS/UPS, Toulouse Cedex 4, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: André
authors.forename: Gérald
authors.initials: G
authors.lastname: Gaibelet
authors.forename: Laurent
authors.initials: L
authors.lastname: Le Guyader
authors.forename: Michèle
authors.initials: M
authors.lastname: Welby
authors.forename: André
authors.initials: A
authors.lastname: Lopez
authors.forename: Chantal
authors.initials: C
authors.lastname: Lebrun
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D004174:Diprenorphine / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D009292:Narcotic Antagonists / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017465:Receptors, Opioid, delta / Q000819:agonists* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism
chemical_list: D008024:Ligands; D009292:Narcotic Antagonists; D009842:Oligopeptides; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D013107:Sphingolipids; C060209:deltorphin II, Ala(2)-; D004174:Diprenorphine; D002784:Cholesterol