title: Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.
abstract: 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.
authors: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors: Rabia U
authors: RU
authors: Malik
authors: Michael
authors: M
authors: Ritt
authors: Brian T
authors: BT
authors: DeVree
authors: Richard R
authors: RR
authors: Neubig
authors: Roger K
authors: RK
authors: Sunahara
authors: Sivaraj
authors: S
authors: Sivaramakrishnan
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008790:Metoprolol / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D055355:Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011412:Propanolamines / Q000494:pharmacology; D011485:Protein Binding; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D000319:Adrenergic beta-Antagonists; D044385:GTP-Binding Protein alpha Subunits; D055355:Opsins; D010446:Peptide Fragments; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C026777:ICI 118551; D000242:Cyclic AMP; D008790:Metoprolol
keywords: (E/D)RY Motif; 7-Helix Receptor; Cell Signaling; Fret; Functional Selectivity; G Protein-coupled Receptors (GPCR); G Proteins
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