Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol

Biochim Biophys Acta. 2008 Jun;1778(6):1483-92. doi: 10.1016/j.bbamem.2008.03.017. Epub 2008 Apr 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cholesterol / metabolism*
  • Diprenorphine / pharmacology*
  • Humans
  • Ligands
  • Membrane Microdomains / genetics
  • Membrane Microdomains / metabolism*
  • Narcotic Antagonists / pharmacology*
  • Oligopeptides / pharmacology*
  • Protein Binding / physiology
  • Protein Structure, Tertiary / physiology
  • Receptors, Opioid, delta / agonists*
  • Receptors, Opioid, delta / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sphingolipids / metabolism

Substances

  • Ligands
  • Narcotic Antagonists
  • Oligopeptides
  • Receptors, Opioid, delta
  • Recombinant Fusion Proteins
  • Sphingolipids
  • deltorphin II, Ala(2)-
  • Diprenorphine
  • Cholesterol