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Publications (2)7.79 Total impact

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    ABSTRACT: Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human micro-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.
    Molecular Membrane Biology 08/2008; 25(5):423-35. · 3.13 Impact Factor
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    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.
    Biochimica et Biophysica Acta 07/2008; 1778(6):1483-92. · 4.66 Impact Factor