Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells

J Biol Chem. 2013 Jun 14;288(24):17167-78. doi: 10.1074/jbc.M113.464065. Epub 2013 Apr 29.


Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the β2-AR (E/D)RY ionic lock enhances interaction with Gs. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of β2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.

Keywords: (E/D)RY Motif; 7-Helix Receptor; Cell Signaling; Fret; Functional Selectivity; G Protein-coupled Receptors (GPCR); G Proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic beta-2 Receptor Agonists / pharmacology
  • Adrenergic beta-Antagonists / pharmacology
  • Amino Acid Motifs
  • Amino Acid Substitution
  • Cyclic AMP / metabolism
  • Fluorescence Resonance Energy Transfer
  • GTP-Binding Protein alpha Subunits / chemistry
  • GTP-Binding Protein alpha Subunits / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • HEK293 Cells
  • Humans
  • Metoprolol / pharmacology
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Opsins / chemistry
  • Opsins / genetics
  • Opsins / metabolism*
  • Peptide Fragments / metabolism
  • Propanolamines / pharmacology
  • Protein Binding
  • Protein Stability / drug effects
  • Protein Structure, Secondary
  • Receptors, Adrenergic, beta-2 / chemistry
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Single-Cell Analysis


  • Adrenergic beta-2 Receptor Agonists
  • Adrenergic beta-Antagonists
  • GTP-Binding Protein alpha Subunits
  • Opsins
  • Peptide Fragments
  • Propanolamines
  • Receptors, Adrenergic, beta-2
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • ICI 118551
  • Cyclic AMP
  • Metoprolol