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

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

Rabia U Malik  1 , Michael Ritt, Brian T DeVree, Richard R Neubig, Roger K Sunahara, Sivaraj Sivaramakrishnan
Affiliations

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

Rabia U Malik et al. J Biol Chem. .

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.

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

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Figures

FIGURE 1.
FIGURE 1.
FRET-based SPASM sensors for opsin and β2-AR are intact and functional. a, schematics of the GPCR-Gα C-terminal peptide sensors (left), sensor in the inactive (middle), and active (right) conformation. Protein domains were separated with Gly-Ser-Gly (GSG)4 linkers to ensure rotational freedom. No-pep sensors do not contain the Gα C terminus peptide. b, opsin-t-mod and β2-AR-s-pep sensor localization to the plasma membrane in HEK293 live cells. c, fluorescence SDS-PAGE gel scans of HEK293 membranes expressing β2-AR no-pep or s-pep sensors. Intact membrane localization is witnessed by distinct 110 kDa bands in fractions containing membrane (P) but not supernatant (S). d, cAMP levels in the presence or absence of agonist (100 μm isoproterenol) for untransfected (gray) or HEK293 cells expressing β2-AR-no-pep sensor (white). Specificity of agonist-stimulated sensor response was verified by suppression with antagonists (150 μm metoprolol or 10 μm ICI 118,551). Results are expressed as mean ± S.E. ***, p < 0.001; n > 10. mCit, mCitrine; mCer, mCerulean; ISO, isoproterenol.
FIGURE 2.
FIGURE 2.
Influence of endogenous Gα levels on sensor FRET measurements. a and b, β2-AR sensors are expressed at least 5-fold in excess of three endogenous Gα subtypes (Gαs/Gαi/Gαq). a, fluorescence SDS-PAGE gel scans of HEK293 membranes expressing β2-AR-Gαs fusion sensor. b, HEK293 membranes expressing the β2-AR-Gα fusion sensors were digested with tobacco etch virus protease to cleave a site between β2-AR-mCitrine and ER/K-α-helix-mCerulean-Gα. Membranes were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with anti-Gαs (sc-823; 1:1000), anti-Gαq antibody (sc-393; 1:1000), or anti-Gαi2 antibody (sc-13534; 1:200). Intact Gα expression is witnessed by distinct 80, 76, and 75 kDa bands for tobacco etch virus-digested Gαs, Gαq, and Gαi2 fusion sensors, respectively. c and d, FRET ratios (mCitrine, 525 nm; mCerulean, 475 nm) of the β2-AR-s-pep sensor co-expressed with unlabeled (dark) Gαs (c) or Gαq (d). Ratio of plasmid DNA of β2-AR-s-pep:Gα used for the transfections is indicated along abscissa (at least 5-fold overexpression of indicated Gα compared with endogenous Gα by densitometry). Bottom panels, immunoblots of membranes transfected with plasmid DNA at indicated ratios probed with anti-Gαs (c) or anti-Gαq antibodies (d). S, supernatant; P, membrane.
FIGURE 3.
FIGURE 3.
Gα C terminus peptide specifically binds to the active conformation of GPCRs in live HEK293 cells. a, schematics of the GPCR-Gα C-terminal peptide sensors (top); crystal structures of β2-AR in the inactive (middle; PDB code 3NY8) and active (bottom; PDB code 3SN6) conformation. The Gαs C terminus (s-pep; red) binds to the active β2-AR conformation induced via stimulation with agonist. b–j, GPCR condition specified at the top left and sensor abbreviation along abscissa. b, change in FRET ratio following agonist (9-cis-retinal + light) treatment for opsin-pep sensors. FRET spectra (mCerulean (mCer) excitation, 430 nm) normalized to mCerulean emission (475 nm) for β2-AR-s-pep (c), β2-AR-i-pep sensors for samples treated with or without agonist (isoproterenol) (d). e and f, change in FRET ratio following agonist (isoproterenol; ISO) treatment for β2-AR-pep sensors. f, dose-dependent inhibition of FRET with inverse agonist (ICI 118,551 (ICI); gray line). g, basal cAMP levels for β2-AR-no-pep sensors expressing the constitutively active β2-AR mutants (CAM, L272A). h, FRET spectra (mCerulean excitation, 430 nm) normalized to mCerulean emission (475 nm) for WT (black) and a constitutively active mutant (CAM; green) β2-AR-s-pep sensor. i, gain in FRET following induction of constitutively active mutations (CAM, L272A) for β2-AR-s-pep sensors. j, scatter plot of individual FRET ratio measurements (open circles) for indicated β2-AR-pep sensors/conditions derived from three independent experiments (colored red, green, and blue), collected on three different days. Results are expressed as mean ± S.E. ***, p < 0.001; n > 18.
FIGURE 4.
FIGURE 4.
Mutagenesis of (E/D)RY motif interactions in β2-AR induces an active conformation. a, crystal structures of β2-AR in the inactive (top; PDB code 3NY8) and active (bottom; PDB code 3SN6) conformation. Top, in the inactive state, the DRY motif residues in β2-AR display electrostatic interactions formed between Arg-131 (blue) and Asp-130/Glu-268 residues (red). Bottom, indicated residues move apart following β2-AR activation. b–e, GPCR/condition specified at the top left, and sensor abbreviations are shown along the abscissa. cAMP levels of HEK293 cells expressing wild-type (no-pep) for the indicated (E/D)RY mutant β2-AR-no-pep sensor in the absence (b) or presence (c) of agonist (100 μm isoproterenol (ISO)). d, FRET ratios (mCitrine/mCerulean, 525 nm/475 nm) of β2-AR (E/D)RY motif single and double (D/E, D130N,E268N) mutant s-pep sensors. e, change in FRET following agonist (100 μm isoproterenol) treatment of (E/D)RY mutant β2-AR-s-pep sensors. f, the affinity for agonist (isoproterenol) was measured for WT, D130N, and R131A β2-AR-no-pep sensors by competitive inhibition of [3H]DHA binding. Results are expressed as percent of radioligand bound in the absence of competitor. g, change in [35S]GTPγS binding induced by 10 μm inverse agonist ICI 118,551 for WT, D130N, and R131A β2-AR-no-pep sensors. h, competitive displacement of [3H]dihydroalprenolol binding by ICI 118,551 for WT, D130N, and R131A β2-AR-no-pep sensors. Results are expressed as mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05; ***, p < 0.001; n > 18.
FIGURE 5.
FIGURE 5.
Opsin-specific interactions centered on residue Lys-296 are both necessary and sufficient to stabilize an inactive conformation. a, electrostatic interactions formed by the (E/D)RY motif are indicated on the crystal structure of inactive, dark rhodopsin (opsin + 9-cis-retinal; PDB code 1GZM). b–f, GPCR condition specified at top left, and sensor abbreviations are specified along abscissa. b and c, FRET ratios (mCitrine/mCerulean, 525 nm/475 nm) of basal (b, untreated) and change in FRET (c) following retinal addition and photo-activation of the (E/D)RY motif mutant t-mod sensors of opsin. d, RP inducing constitutively active opsin mutations and their interactions are indicated in the inactive dark rhodopsin crystal structure (PDB code 1GZM). e, FRET ratios of RP mutant opsin-t-mod sensors in the absence of retinal. f, change in FRET following retinal addition and photoactivation of sensors in e. Results are expressed as mean ± S.E. **, p < 0.01; ***, p < 0.001; n > 18.
FIGURE 6.
FIGURE 6.
Inverse agonist ICI 118,551 requires a functional (E/D)RY motif to suppress β2-AR basal activity. a, electrostatic interactions formed by the (E/D)RY motif are indicated on the crystal structure of β2-AR bound to inverse agonist (ICI 118,551; ICI) (PDB code 3NY8). b, change in FRET following inverse agonists (10 μm ICI 118,551 or 150 μm metoprolol (Meto)) treatment of indicated (E/D)RY motif mutant β2-AR-s-pep sensors. c, ICI 118,551 induced percent cAMP inhibition of HEK293 cells expressing wild-type (no-pep) or the indicated (E/D)RY motif mutant β2-AR-no-pep sensors. Results are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n > 18.
FIGURE 7.
FIGURE 7.
Sensors detect stabilization of Gi conformations in β2-AR stimulated with metoprolol. Shown is the change in FRET following treatment of indicated β2-AR-pep sensors with inverse agonist (150 μm metoprolol (Meto)) (a) or with varying concentrations of metoprolol (b). c, percent inhibition of 100 μm isoproterenol (ISO) or 10 μm forskolin-induced (Fsk) cAMP levels with 150 μm metoprolol or 10 μm ICI 118,551, in PTX-treated or untreated HEK293T cells expressing β2-AR-no-pep sensor. Results are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n >18.
FIGURE 8.
FIGURE 8.
Distinct structural mechanisms of β2-AR agonists and inverse agonists. In the absence of ligand (basal state), only a small proportion of the β2-AR population adopts Gs conformations. Isoproterenol (ISO; agonist) treatment destabilizes the DRY ionic lock and enhances interaction with the Gαs C terminus, resulting in activation of adenylyl cyclase. Conversely, ICI 118,551 (ICI; inverse agonist), reinforces the DRY ionic lock, and shifts the equilibrium toward inactive conformations. Biased agonist (metoprolol; Meto) stabilizes Gi conformations, promoting Gi-dependent inhibition of adenylyl cyclase. mCit, mCitrine; mCer, mCerulean.
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