Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles

Biochemistry. 2016 Jan 12;55(1):38-48. doi: 10.1021/acs.biochem.5b01040. Epub 2015 Dec 24.

Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cricetulus
  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / isolation & purification*
  • GTP-Binding Proteins / metabolism
  • Humans
  • Lipids / chemistry*
  • Liposomes / chemistry*
  • Maleates / chemistry*
  • Models, Molecular
  • Nanostructures / chemistry*
  • Pichia / chemistry
  • Pichia / metabolism
  • Polystyrenes / chemistry*
  • Receptors, Ghrelin / chemistry
  • Receptors, Ghrelin / isolation & purification
  • Receptors, Ghrelin / metabolism
  • Receptors, Melatonin / chemistry
  • Receptors, Melatonin / isolation & purification
  • Receptors, Melatonin / metabolism
  • Solubility

Substances

  • Lipids
  • Liposomes
  • Maleates
  • Polystyrenes
  • Receptors, Ghrelin
  • Receptors, Melatonin
  • styrene-maleic acid polymer
  • GTP-Binding Proteins