Allosteric coupling from G protein to the agonist-binding pocket in GPCRs

Journal name:
Nature
Volume:
535,
Pages:
182–186
Date published:
DOI:
doi:10.1038/nature18324
Received
Accepted
Published online

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other1. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades1. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins2. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

At a glance

Figures

  1. Guanine nucleotides influence antagonist binding to β2AR•Gs complexes.
    Figure 1: Guanine nucleotides influence antagonist binding to β2AR•Gs complexes.

    a, Binding of 2 nM [3H]DHAP to β2AR•Gs in the absence or presence of GDP. Addition of apyrase to GDP-bound β2AR•Gs led to a progressive decrease in [3H]DHAP binding over time, which could be restored with excess GDP. b, Addition of increasing concentrations of GDP enhances the rate and extent of [3H]DHAP binding to apyrase-treated β2AR•Gs complexes. a, Data are shown as mean ± standard error of the mean (s.e.m.) from n = 3 independent experiments performed in duplicate. b, Data are representative of three independent experiments.

  2. Trapping active-state β2AR with Nb80 slows both antagonist and agonist association.
    Figure 2: Trapping active-state β2AR with Nb80 slows both antagonist and agonist association.

    a, Nb80 (red) mimics G protein (yellow) in both its binding site and the β2AR conformation it stabilizes. The structure of Nb80-bound β2AR (Protein Data Bank (PDB) accession 3P0G) is shown in orange, Gs-bound β2AR (PDB accession 3SN6) in cyan. b, Pre-incubation of the β2AR with increasing concentrations of Nb80 progressively slows association of neutral antagonist [3H]DHAP to the β2AR. ce, Nb80 also slows association of full agonist [3H]formoterol (c), partial agonist [3H]CGP12177 (d), and inverse agonist [3H]carvedilol (e) to the β2AR. f, Nb80 stabilizes the closed, active conformation and slows [3H]DHAP dissociation from the β2AR in a concentration-dependent manner. b, f, Data are representative of three independent experiments. All other data are specific binding, shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  3. Activation of the β2AR closes the hormone-binding site.
    Figure 3: Activation of the β2AR closes the hormone-binding site.

    a, Stabilization of the β2AR active conformation by Gs (or Nb80) brings the side chains of Phe193ECL2 and Tyr3087.35 closer to one another compared to their positions in structures in the absence of G protein. b, Closer view of the orthosteric site, highlighting Phe193ECL2 and Tyr3087.35. Distances (in Å) between the hydroxyl on Tyr3087.35 and 2-carbon on the phenyl ring of Phe193ECL2 are indicated. c, d, A surface view comparing the extracellular face of β2AR in inactive (c) or active (d) conformations, showing how G-protein-stabilized structural rearrangements occlude the hormone-binding site in the active state. e, f, Cutaway view illustrating closure of the hormone-binding site around the bound agonist in the active state. The inverse agonist carazolol is shown in orange, the agonist BI-167107 is shown in yellow.

  4. Allosteric communication between the β2AR G-protein- and hormone-binding sites.
    Figure 4: Allosteric communication between the β2AR G-protein- and hormone-binding sites.

    a, In the β2AR active state (cyan), the cytoplasmic end of TM6 moves away from the receptor core by ~14 Å relative to its position in the inactive-state structure, allowing for an inward movement of TM7. b, Rotation of TM7 allows Tyr3267.53 (of the highly conserved NPxxY motif) to fill the space vacated by the conserved aliphatic residue Ile2786.40. c, The rotation of TM7 repositions Tyr3087.35 and Lys3057.32. This conformational change allows Lys3057.32 to coordinate the backbone carbonyl of Phe193ECL2, stabilizing its movement towards Tyr3087.35 to form a lid over the hormone-binding site.

  5. Basis for G-protein-dependent high-affinity agonist binding.
    Figure 5: Basis for G-protein-dependent high-affinity agonist binding.

    Agonist binding promotes the G-protein–receptor (R) interaction and GDP release from the G-protein heterotrimer (Gα (α) Gβγ (βγ)). In this nucleotide-free state, the C-terminal helix of Gα remains embedded in the receptor core, stabilizing the conformational changes at both the intracellular and extracellular faces of the receptor. At the extracellular side, the orthosteric ligand-binding site closes around the bound agonist, sterically opposing agonist dissociation and thereby enhancing the observed affinity. Constitutive (basal) receptor activity may also activate the G protein, releasing GDP and thereby stabilizing the closed conformation of the receptor in the absence of an agonist. See also Extended Data Fig. 10.

  6. Confirmation of nucleotide removal from β2AR•Gs by apyrase.
    Extended Data Fig. 1: Confirmation of nucleotide removal from β2AR•Gs by apyrase.

    Gs and Flag-tagged β2AR were reconstituted in rHDL and treated with the non-specific nucleotide lyase, apyrase. Samples were applied to an anti-Flag affinity resin to remove products of the GDP degradation (GMP and Pi). Samples were incubated with 100 nM [35S]GTPγS at room temperature. At various times, samples were subjected to rapid filtration through glass fibre filters (GF/B) followed by 10 volumes of ice-cold buffer washes containing 10 μM GDP. Filters were dried and subjected to liquid scintillation counting (Top-Count, Perkin-Elmer). To a first approximation, the rapid binding event suggests that the complex is empty of nucleotide, based on the limited temporal resolution of this mixing and filtration technique. [3H]DHAP and [35S]GTPγS binding to the reconstituted complex yields a final R:G ratio of 1:0.95, suggesting that up to 95% of the β2AR–rHDL particles contain a single functional G protein. This suggests that only those G proteins associated with the β2AR will bind [35S]GTPγS within this time frame in the absence of receptor agonists. Data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  7. GDP accelerates [3H]DHAP binding to β2AR•Gs.
    Extended Data Fig. 2: GDP accelerates [3H]DHAP binding to β2AR•Gs.

    a, Time course monitoring [3H]DHAP association to apyrase-treated β2AR•Gs complexes in the presence of varying GDP concentrations. GDP increases both the observed association rate constant and the maximum binding of [3H]DHAP. b, Concentration–response curve showing enhancement of the observed [3H]DHAP association rate constant by GDP (half-maximum effective concentration (EC50) = 181 ± 66 nM). All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  8. Effect of guanine nucleotides on [3H]DHAP binding to β2AR•Gs.
    Extended Data Fig. 3: Effect of guanine nucleotides on [3H]DHAP binding to β2AR•Gs.

    a, In saturation binding assays, addition of GTPγS to apyrase-treated β2AR•Gs complexes increased the observed maximal binding, Bmax, for [3H]DHAP without significantly altering Kd (control: Bmax = 5.5 ± 0.52 fmol, Kd = 0.88 nM; +GTPγS: Bmax = 16.6 ± 1.9 fmol, Kd = 0.56 nM). b, Both GDP and GTPγS could enhance maximal [3H]DHAP binding in a concentration-dependent manner (GDP log(EC50) = −6.42 ± 0.12, or EC50 ≈ 386 nM; GTPγS log(EC50) = −7.45 ± −0.16, or EC50 ≈ 35 nM). All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  9. Effect of Nb80 on antagonist binding to the β2AR.
    Extended Data Fig. 4: Effect of Nb80 on antagonist binding to the β2AR.

    a, Association of [3H]DHAP is progressively slowed after pre-incubation of the β2AR with increasing concentrations of Nb80. b, If [3H]DHAP is allowed to first equilibrate with the β2AR, Nb80 slows [3H]DHAP dissociation from β2AR in a concentration-dependent manner. c, Owing to the dramatic slowing of [3H]DHAP binding kinetics, Nb80 (but not a control nanobody, Nb30, which has no effect on agonist affinity for β2AR) seems competitive with [3H]DHAP if insufficient time is given to reach equilibrium. Data shown are from assays incubated for 90 min at room temperature. All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  10. Y308A mutation abolishes the rate-slowing effects of Nb80.
    Extended Data Fig. 5: Y308A mutation abolishes the rate-slowing effects of Nb80.

    a, b, Time course of [3H]DHAP binding to wild-type (WT) β2AR (a) or β2AR(Y308A) (b) after pre-incubation of receptor with Nb80. Nb80 significantly slowed [3H]DHAP association to wild-type β2AR (−Nb80 observed rate constant, kobs = 0.45 ± 0.05 min−1 or association half-time, t½ = 1.5 ± 0.2 min, +Nb80 kobs = 0.20 ± 0.03 min−1 or t½ = 3.5 ± 0.5 min; P = 0.011 by an unpaired two-tailed t-test), but less effectively slowed [3H]DHAP association to β2AR(Y308A) (−Nb80 kobs = 0.50 ± 0.06 min−1 or t½ = 1.4 ± 0.2 min; +Nb80 kobs = 0.32 ± 0.01 min−1 or t½ = 2.2 ± 0.1 min; P = 0.05 by an unpaired two-tailed t-test. All data are shown as mean ± s.e.m. from n = 4 (−Nb80) or n = 3 (+Nb80) independent experiments performed in duplicate. c, d, Time course of [3H]formoterol binding to wild-type β2AR (c) or β2AR(Y308A) (d) after pre-incubation of receptor with Nb80. Nb80 slowed [3H]formoterol association to wild-type β2AR (0.1 μM Nb80 kobs = 0.68 ± 0.13 min−1 or t½ = 1.0 ± 0.2 min, 10 μM Nb80 kobs = 0.27 ± 0.05 min−1 or t½ = 2.6 ± 0.5 min; P = 0.031 by an unpaired two-tailed t-test). However, with β2AR(Y308A), Nb80 had little effect on the observed association rate constant but enhanced the amount of [3H]formoterol bound (0.1 μM Nb80 kobs = 0.37 ± 0.11 min−1 or t½ = 1.9 ± 0.6 min with a plateau of 10.1 ± 0.8 fmol, 10 μM Nb80 kobs = 0.53 ± 0.13 min−1 or t½ = 1.3 ± 0.4 min with a plateau of 21.3 ± 1.2 fmol; unpaired two-tailed t-test of the kobs values showed P = 0.4). All data are shown as mean ± s.e.m. from n = 4 independent experiments performed in duplicate.

  11. The closed conformation stabilized by agonist and G protein (or mimic).
    Extended Data Fig. 6: The closed conformation stabilized by agonist and G protein (or mimic).

    Illustrated are the crystal structures of agonist-versus inverse-agonist-bound β2AR (cyan) and β1AR (yellow), where only β2AR is bound to G protein. Similarly, the MOPr (orange) adopts a closed conformation upon binding the G-protein surrogate, Nb39. β2AR, PDB accession 2RH1; β2AR•Gs, PDB accession 3SN6; β1AR, PDB accession 2YCW; β1AR-iso, PDB accession 2Y03; MOPr, PDB accession 4DKL; MOPr–Nb39, PDB accession 5C1M; M2R, PDB accession 3UON; M2R–Nb9-8, PDB accession 4MQS.

  12. Effect of guanine nucleotides on [3H]antagonist binding are also seen in competition binding assays.
    Extended Data Fig. 7: Effect of guanine nucleotides on [3H]antagonist binding are also seen in competition binding assays.

    a, Agonist (isoproterenol) competition binding using apyrase-treated β2AR•Gs complexes shows the characteristic G-protein-dependent shift in agonist affinity, along with a dramatic increase in total [3H]DHAP binding, upon the addition of 10 μM GTPγS. b, Normalization of the data from a yields a plot representative of what is commonly reported in the literature. c, Similar to the β2AR, agonist (morphine) competition binding using MOPr•Go complexes shows the characteristic G-protein-dependent shift in agonist affinity, along with a dramatic increase in total [3H]DPN binding, upon the addition of 10 μM GTPγS. d, Normalization of the data from c. All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  13. The MOPr and M2R behave similarly to the β2AR when bound to nucleotide-free G protein or an active-state-stabilizing nanobody.
    Extended Data Fig. 8: The MOPr and M2R behave similarly to the β2AR when bound to nucleotide-free G protein or an active-state-stabilizing nanobody.

    a, After apyrase treatment of M2R•Go complexes, addition of 10 μM GTPγS enhances association of [3H]N-methylscopolamine ([3H]NMS) to M2R (vehicle kobs = 0.32 ± 0.02 min−1 or t½ = 2.2 ± 0.1 min, +GTPγS kobs = 0.54 ± 0.02 min−1 or t½ = 1.3 ± 0.1 min; P = 0.002 by an unpaired two-tailed t-test). Data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate. Addition of GDP was also able to increase the rate of [3H]NMS binding (inset; log (EC50) = 6.91 ± 0.18 or EC50 ≈ 123 nM; mean ± s.e.m. from n = 2 independent experiments performed in duplicate). b, Pre-treatment of M2R with either 10 μM (black circles) or 100 μM (red squares) Nb9-8 (ref. 27) impairs association of [3H]iperoxo to M2R (10 μM Nb9-8 kobs = 0.68 ± 0.09 min−1 or t½ = 1.0 ± 0.2 min, 100 μM Nb9-8 kobs = 0.25 ± 0.04 min−1 or t½ = 2.8 ± 0.5 min; P = 0.04 by an unpaired two-tailed t-test). Data are shown as mean ± s.e.m. from n = 3 (10 μM Nb9-8) or n = 2 (100 μM Nb9-8) independent experiments performed in duplicate. c, Addition of 10 μM GTPγS to apyrase-treated MOPr•Go complexes hastened association of the antagonist [3H]diprenorphine ([3H]DPN) to MOPr (apyrase kobs = 0.06 ± 0.02 min−1 or t½ = 9.8 ± 1.3 min, +GTPγS kobs = 0.12 ± 0.01 min−1 or t½ = 5.6 ± 0.6 min; P = 0.1 by an unpaired two-tailed t-test). The effect of nucleotide-free G protein was recapitulated by pre-incubating MOPr with Nb39 (ref. 28) (inset; control kobs = 0.13 ± 0.01 min−1, +100 μM Nb39 kobs = 0.07 ± 0.02 min−1). Data are shown as mean ± s.e.m. from n = 2 (MOPr•Go) or n = 3 (MOPr + Nb39) independent experiments performed in duplicate.

  14. The extracellular regions in the active conformations of peptide hormone/agonist receptors MOPr and NTS-R1.
    Extended Data Fig. 9: The extracellular regions in the active conformations of peptide hormone/agonist receptors MOPr and NTS-R1.

    Illustrated are the crystal structures of the inactive and active (or partially active neurotensin receptor 1, NTS-R1) conformations of the MOPr and NTS-R1 from the top or extracellular view of the receptor. The surface rendering highlights residues or structure on the extracellular face that change upon receptor activation (circled). The MOPr in its inactive conformation (purple) is compared to the Nb39-bound (G-protein mimic) form in blue. Similarly, the inactive NTS-R1 (ref. 33) (green) is compared with a mutant NTS-R1 (ref. 34) that adopts a partially active conformation (orange). MOPr, PDB accession 4DKL; MOPr-Nb39, PDB accession 5C1M; NTS-R1, PDB accession 4GRV; active-like NTS-R1, PDB accession 4XEE.

  15. Model of G-protein-dependent high-affinity agonist binding.
    Extended Data Fig. 10: Model of G-protein-dependent high-affinity agonist binding.

    a, b, As in Fig. 5, nucleotide-free G-protein-stabilized family A GPCRs experience alterations in the extracellular face of the receptor, thus affecting the orthosteric-binding site. In a monoamine receptor such as the β2AR, G-protein binding and GDP loss accompanies the stabilization of a closed, active conformation of the receptor, as in a. b, For family members such as MOPr or NTS-R1, where the peptide hormones/agonists are considerably larger, the influence of the G-protein-mediated changes in the extracellular domain structure result in similar effects on orthosteric ligand dissociation. Rather than closing over the orthosteric site as with monoamine receptors as in a, the extracellular face may contain structures and residues that ‘pinch’ the larger ligands.

Videos

  1. Activation of the 2AR
    Video 1: Activation of the β2AR
    Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). For reference, epinephrine is modeled in the orthosteric binding site. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC). Highlighted are Phe193ECL2 and Tyr3087.35.
  2. Comparison of 1AR and 2AR
    Video 2: Comparison of β1AR and β2AR
    The closed, active conformation is stabilized by the G protein. Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (cyan, PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). Superimposed on top is a similar morph transitioning between the carazolol-boundββ1-adrenergic receptor (β1AR, lime green, PDB: 2YCW) and isoproterenol-bound (but not G protein- or Nb-bound, PDB: 2Y03). While the β2AR adopts a closed conformation stabilized by agonist and G protein, the β1AR bound only to isoproterenol does not. For reference, epinephrine is modeled in the orthosteric binding site. Note that nanobody Nb80 has been omitted from the animation for simplicity. Highlighted are Phe193ECL2 and Tyr3087.35 on β2AR and the conserved residues Phe201ECL2 and Phe3527.35 on β1AR. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  3. Activation of the μ-opioid receptor, MOPr
    Video 3: Activation of the μ-opioid receptor, MOPr
    The ribbon structure of the μ-opioid receptor (MOPr) in its inactive conformation bound to β-funaltrexamine (cyan, PDB: 4DKL) and the MOPr in its active conformation bound to agonist BU72 and Nb39 (orange, PDB: 5C1M). For reference only BU72 is displayed as spheres. Note that Nb39 is not illustrated for simplicity. Morph between the inactive and active conformations was generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  4. Activation of the M2 muscarinic receptor, M2R
    Video 4: Activation of the M2 muscarinic receptor, M2R
    Top view of the ribbon structure of the M2 muscarinic receptor (M2R) in its inactive conformation bound to antagonist 3-quinuclidinyl benzilate, (QNB, PDB: 3UON) and the M2R in its active conformation bound to agonist iperoxo and nanobody Nb9-8 (PDB: 4MQS). Illustrated are sidechain residues Y1043.33, Y4036.38 and Y4267.39 to highlight the ‘lid-like’ structure over the orthosteric site. Acetylcholine is modeled into the iperoxo binding site and illustrated in stick figure for reference purposes. Note that Nb9-8 is not depicted for simplicity. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  5. Activation of the M2R side view
    Video 5: Activation of the M2R side view
    Side view of the ribbon structure of the M2R (above) to highlight the ‘lid-like’ structure over the orthosteric site. Illustrated are side chain residues Y4036.38 and Y4267.39 moving toward Y1043.33 during the formation of the active conformation. Note that TM5 was removed from the rendering so that the tyrosine residues may be easily viewed. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  6. Stabilization of the active state of rhodopsin by G protein
    Video 6: Stabilization of the active state of rhodopsin by G protein
    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and the photoactivated meta-stable form of rhodopsin bound to the C-terminal fragment of the G protein alpha subunit, transducin (transducin not shown) (PDB:3PAR) (Choe H. W. et al. Crystal structure of metarhodopsin II. Nature471, 651–655 (2011)). Note the pre-existing ‘lid-like’ structure over the orthosteric site formed by the ECL2 and N-terminus. Photoisomerization of 11-cis retinal is illustrated in magenta. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  7. Stabilization of the active state of rhodopsin by arrestin
    Video 7: Stabilization of the active state of rhodopsin by arrestin
    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and an active mutant of opsin bound to activated arrestin (arrestin not shown) (PDB:4ZWJ) (Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature523, 561-567 (2015)). Note the similarities in conformational changes as with metarhodopsin bound to the C-terminal helix of transducin. Photoisomerization of 11-cis retinal, based on the metarhodopsin structures (Extended data: Movie SM6) has been modeled into the opsin-arrestin structure and is illustrated in magenta. Morphs were generated between inactive and arrestin-bound conformations of rhodopsin using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12.) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

References

  1. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nature Rev. Mol. Cell Biol. 3, 639650 (2002)
  2. Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549555 (2011)
  3. Venter, J. C. et al. The sequence of the human genome. Science 291, 13041351 (2001)
  4. Sprang, S. R. G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639678 (1997)
  5. Chung, K. Y. et al. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477, 611615 (2011)
  6. Westfield, G. H. et al. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl Acad. Sci. USA 108, 1608616091 (2011)
  7. Maguire, M. E., Van Arsdale, P. M. & Gilman, A. G. An agonist-specific effect of guanine nucleotides on binding to the beta adrenergic receptor. Mol. Pharmacol. 12, 335339 (1976)
  8. Ross, E. M., Maguire, M. E., Sturgill, T. W., Biltonen, R. L. & Gilman, A. G. Relationship between the β-adrenergic receptor and adenylate cyclase. J. Biol. Chem. 252, 57615775 (1977)
  9. De Lean, A., Stadel, J. M. & Lefkowitz, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol. Chem. 255, 71087117 (1980)
  10. Yao, X. J. et al. The effect of ligand efficacy on the formation and stability of a GPCR–G protein complex. Proc. Natl Acad. Sci. USA 106, 95019506 (2009)
  11. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175180 (2011)
  12. Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534538 (2013)
  13. Lefkowitz, R. J. & Williams, L. T. Catecholamine binding to the β-adrenergic receptor. Proc. Natl Acad. Sci. USA 74, 515519 (1977)
  14. Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575579 (2013)
  15. Bokoch, M. P. et al. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463, 108112 (2010)
  16. Kikkawa, H., Isogaya, M., Nagao, T. & Kurose, H. The role of the seventh transmembrane region in high affinity binding of a β2-selective agonist TA-2005. Mol. Pharmacol. 53, 128134 (1998)
  17. Dror, R. O. et al. Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc. Natl Acad. Sci. USA 108, 1311813123 (2011)
  18. Rosenbaum, D. M. et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature 469, 236240 (2011)
  19. Warne, T. et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241244 (2011)
  20. Samama, P., Cotecchia, S., Costa, T. & Lefkowitz, R. J. A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 46254636 (1993)
  21. Weiss, J. M., Morgan, P. H., Lutz, M. W. & Kenakin, T. P. The cubic ternary complex receptor-occupancy model. I. Model description. J. Theor. Biol. 178, 151167 (1996)
  22. Burgisser, E., De Lean, A. & Lefkowitz, R. J. Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotide. Proc. Natl Acad. Sci. USA 79, 17321736 (1982)
  23. Bylund, D. B., Gerety, M. E., Happe, H. K. & Murrin, L. C. A robust GTP-induced shift in α2-adrenoceptor agonist affinity in tissue sections from rat brain. J. Neurosci. Methods 105, 159166 (2001)
  24. Prater, M. R., Taylor, H., Munshi, R. & Linden, J. Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine. Mol. Pharmacol. 42, 765772 (1992)
  25. Werling, L. L., Puttfarcken, P. S. & Cox, B. M. Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. Mol. Pharmacol. 33, 423431 (1988)
  26. Haga, K. et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547551 (2012)
  27. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101106 (2013)
  28. Manglik, A. et al. Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature 485, 321326 (2012)
  29. Huang, W. et al. Structural insights into μ-opioid receptor activation. Nature 524, 315321 (2015)
  30. Katritch, V. et al. Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39, 233244 (2014)
  31. Kozasa, T. & Gilman, A. G. Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of α12 and inhibition of adenylyl cyclase by αz. J. Biol. Chem. 270, 17341741 (1995)
  32. Whorton, M. R. et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl Acad. Sci. USA 104, 76827687 (2007)
  33. White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508513 (2012)
  34. Krumm, B. E., White, J. F., Shah, P. & Grisshammer, R. Structural prerequisites for G-protein activation by the neurotensin receptor. Nature Commun. 6, 78957895 (2015)

Download references

Author information

  1. These authors contributed equally to this work.

    • Brian T. DeVree &
    • Jacob P. Mahoney

Affiliations

  1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA

    • Brian T. DeVree,
    • Jacob P. Mahoney,
    • Gisselle A. Vélez-Ruiz,
    • Adam J. Kuszak,
    • Elin Edwald &
    • Roger K. Sunahara
  2. Department of Cellular and Molecular Physiology, Stanford University, Palo Alto, California 94305, USA

    • Soren G. F. Rasmussen,
    • Juan-Jose Fung,
    • Aashish Manglik,
    • Matthieu Masureel,
    • Yang Du,
    • Rachel A. Matt &
    • Brian K. Kobilka
  3. Structural Biology Research Center, VIB, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium

    • Els Pardon
  4. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium

    • Jan Steyaert
  5. Department of Pharmacology, University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA

    • Roger K. Sunahara

Contributions

B.T.D., J.P.M., G.A.V.-R., B.K.K. and R.K.S. designed the experiments. B.T.D., J.P.M., G.A.V.-R. and A.J.K. performed research; S.G.F.R., E.E., J.-J.F., A.M., M.M., Y.D., R.A.M., E.P. and J.S. contributed valuable reagents/analytic tools; B.T.D., J.P.M., G.A.V.-R., B.K.K. and R.K.S. analysed data; and B.T.D., J.P.M., B.K.K. and R.K.S. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Confirmation of nucleotide removal from β2AR•Gs by apyrase. (100 KB)

    Gs and Flag-tagged β2AR were reconstituted in rHDL and treated with the non-specific nucleotide lyase, apyrase. Samples were applied to an anti-Flag affinity resin to remove products of the GDP degradation (GMP and Pi). Samples were incubated with 100 nM [35S]GTPγS at room temperature. At various times, samples were subjected to rapid filtration through glass fibre filters (GF/B) followed by 10 volumes of ice-cold buffer washes containing 10 μM GDP. Filters were dried and subjected to liquid scintillation counting (Top-Count, Perkin-Elmer). To a first approximation, the rapid binding event suggests that the complex is empty of nucleotide, based on the limited temporal resolution of this mixing and filtration technique. [3H]DHAP and [35S]GTPγS binding to the reconstituted complex yields a final R:G ratio of 1:0.95, suggesting that up to 95% of the β2AR–rHDL particles contain a single functional G protein. This suggests that only those G proteins associated with the β2AR will bind [35S]GTPγS within this time frame in the absence of receptor agonists. Data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  2. Extended Data Figure 2: GDP accelerates [3H]DHAP binding to β2AR•Gs. (310 KB)

    a, Time course monitoring [3H]DHAP association to apyrase-treated β2AR•Gs complexes in the presence of varying GDP concentrations. GDP increases both the observed association rate constant and the maximum binding of [3H]DHAP. b, Concentration–response curve showing enhancement of the observed [3H]DHAP association rate constant by GDP (half-maximum effective concentration (EC50) = 181 ± 66 nM). All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  3. Extended Data Figure 3: Effect of guanine nucleotides on [3H]DHAP binding to β2AR•Gs. (167 KB)

    a, In saturation binding assays, addition of GTPγS to apyrase-treated β2AR•Gs complexes increased the observed maximal binding, Bmax, for [3H]DHAP without significantly altering Kd (control: Bmax = 5.5 ± 0.52 fmol, Kd = 0.88 nM; +GTPγS: Bmax = 16.6 ± 1.9 fmol, Kd = 0.56 nM). b, Both GDP and GTPγS could enhance maximal [3H]DHAP binding in a concentration-dependent manner (GDP log(EC50) = −6.42 ± 0.12, or EC50 ≈ 386 nM; GTPγS log(EC50) = −7.45 ± −0.16, or EC50 ≈ 35 nM). All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  4. Extended Data Figure 4: Effect of Nb80 on antagonist binding to the β2AR. (275 KB)

    a, Association of [3H]DHAP is progressively slowed after pre-incubation of the β2AR with increasing concentrations of Nb80. b, If [3H]DHAP is allowed to first equilibrate with the β2AR, Nb80 slows [3H]DHAP dissociation from β2AR in a concentration-dependent manner. c, Owing to the dramatic slowing of [3H]DHAP binding kinetics, Nb80 (but not a control nanobody, Nb30, which has no effect on agonist affinity for β2AR) seems competitive with [3H]DHAP if insufficient time is given to reach equilibrium. Data shown are from assays incubated for 90 min at room temperature. All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  5. Extended Data Figure 5: Y308A mutation abolishes the rate-slowing effects of Nb80. (290 KB)

    a, b, Time course of [3H]DHAP binding to wild-type (WT) β2AR (a) or β2AR(Y308A) (b) after pre-incubation of receptor with Nb80. Nb80 significantly slowed [3H]DHAP association to wild-type β2AR (−Nb80 observed rate constant, kobs = 0.45 ± 0.05 min−1 or association half-time, t½ = 1.5 ± 0.2 min, +Nb80 kobs = 0.20 ± 0.03 min−1 or t½ = 3.5 ± 0.5 min; P = 0.011 by an unpaired two-tailed t-test), but less effectively slowed [3H]DHAP association to β2AR(Y308A) (−Nb80 kobs = 0.50 ± 0.06 min−1 or t½ = 1.4 ± 0.2 min; +Nb80 kobs = 0.32 ± 0.01 min−1 or t½ = 2.2 ± 0.1 min; P = 0.05 by an unpaired two-tailed t-test. All data are shown as mean ± s.e.m. from n = 4 (−Nb80) or n = 3 (+Nb80) independent experiments performed in duplicate. c, d, Time course of [3H]formoterol binding to wild-type β2AR (c) or β2AR(Y308A) (d) after pre-incubation of receptor with Nb80. Nb80 slowed [3H]formoterol association to wild-type β2AR (0.1 μM Nb80 kobs = 0.68 ± 0.13 min−1 or t½ = 1.0 ± 0.2 min, 10 μM Nb80 kobs = 0.27 ± 0.05 min−1 or t½ = 2.6 ± 0.5 min; P = 0.031 by an unpaired two-tailed t-test). However, with β2AR(Y308A), Nb80 had little effect on the observed association rate constant but enhanced the amount of [3H]formoterol bound (0.1 μM Nb80 kobs = 0.37 ± 0.11 min−1 or t½ = 1.9 ± 0.6 min with a plateau of 10.1 ± 0.8 fmol, 10 μM Nb80 kobs = 0.53 ± 0.13 min−1 or t½ = 1.3 ± 0.4 min with a plateau of 21.3 ± 1.2 fmol; unpaired two-tailed t-test of the kobs values showed P = 0.4). All data are shown as mean ± s.e.m. from n = 4 independent experiments performed in duplicate.

  6. Extended Data Figure 6: The closed conformation stabilized by agonist and G protein (or mimic). (436 KB)

    Illustrated are the crystal structures of agonist-versus inverse-agonist-bound β2AR (cyan) and β1AR (yellow), where only β2AR is bound to G protein. Similarly, the MOPr (orange) adopts a closed conformation upon binding the G-protein surrogate, Nb39. β2AR, PDB accession 2RH1; β2AR•Gs, PDB accession 3SN6; β1AR, PDB accession 2YCW; β1AR-iso, PDB accession 2Y03; MOPr, PDB accession 4DKL; MOPr–Nb39, PDB accession 5C1M; M2R, PDB accession 3UON; M2R–Nb9-8, PDB accession 4MQS.

  7. Extended Data Figure 7: Effect of guanine nucleotides on [3H]antagonist binding are also seen in competition binding assays. (249 KB)

    a, Agonist (isoproterenol) competition binding using apyrase-treated β2AR•Gs complexes shows the characteristic G-protein-dependent shift in agonist affinity, along with a dramatic increase in total [3H]DHAP binding, upon the addition of 10 μM GTPγS. b, Normalization of the data from a yields a plot representative of what is commonly reported in the literature. c, Similar to the β2AR, agonist (morphine) competition binding using MOPr•Go complexes shows the characteristic G-protein-dependent shift in agonist affinity, along with a dramatic increase in total [3H]DPN binding, upon the addition of 10 μM GTPγS. d, Normalization of the data from c. All data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate.

  8. Extended Data Figure 8: The MOPr and M2R behave similarly to the β2AR when bound to nucleotide-free G protein or an active-state-stabilizing nanobody. (365 KB)

    a, After apyrase treatment of M2R•Go complexes, addition of 10 μM GTPγS enhances association of [3H]N-methylscopolamine ([3H]NMS) to M2R (vehicle kobs = 0.32 ± 0.02 min−1 or t½ = 2.2 ± 0.1 min, +GTPγS kobs = 0.54 ± 0.02 min−1 or t½ = 1.3 ± 0.1 min; P = 0.002 by an unpaired two-tailed t-test). Data are shown as mean ± s.e.m. from n = 3 independent experiments performed in duplicate. Addition of GDP was also able to increase the rate of [3H]NMS binding (inset; log (EC50) = 6.91 ± 0.18 or EC50 ≈ 123 nM; mean ± s.e.m. from n = 2 independent experiments performed in duplicate). b, Pre-treatment of M2R with either 10 μM (black circles) or 100 μM (red squares) Nb9-8 (ref. 27) impairs association of [3H]iperoxo to M2R (10 μM Nb9-8 kobs = 0.68 ± 0.09 min−1 or t½ = 1.0 ± 0.2 min, 100 μM Nb9-8 kobs = 0.25 ± 0.04 min−1 or t½ = 2.8 ± 0.5 min; P = 0.04 by an unpaired two-tailed t-test). Data are shown as mean ± s.e.m. from n = 3 (10 μM Nb9-8) or n = 2 (100 μM Nb9-8) independent experiments performed in duplicate. c, Addition of 10 μM GTPγS to apyrase-treated MOPr•Go complexes hastened association of the antagonist [3H]diprenorphine ([3H]DPN) to MOPr (apyrase kobs = 0.06 ± 0.02 min−1 or t½ = 9.8 ± 1.3 min, +GTPγS kobs = 0.12 ± 0.01 min−1 or t½ = 5.6 ± 0.6 min; P = 0.1 by an unpaired two-tailed t-test). The effect of nucleotide-free G protein was recapitulated by pre-incubating MOPr with Nb39 (ref. 28) (inset; control kobs = 0.13 ± 0.01 min−1, +100 μM Nb39 kobs = 0.07 ± 0.02 min−1). Data are shown as mean ± s.e.m. from n = 2 (MOPr•Go) or n = 3 (MOPr + Nb39) independent experiments performed in duplicate.

  9. Extended Data Figure 9: The extracellular regions in the active conformations of peptide hormone/agonist receptors MOPr and NTS-R1. (553 KB)

    Illustrated are the crystal structures of the inactive and active (or partially active neurotensin receptor 1, NTS-R1) conformations of the MOPr and NTS-R1 from the top or extracellular view of the receptor. The surface rendering highlights residues or structure on the extracellular face that change upon receptor activation (circled). The MOPr in its inactive conformation (purple) is compared to the Nb39-bound (G-protein mimic) form in blue. Similarly, the inactive NTS-R1 (ref. 33) (green) is compared with a mutant NTS-R1 (ref. 34) that adopts a partially active conformation (orange). MOPr, PDB accession 4DKL; MOPr-Nb39, PDB accession 5C1M; NTS-R1, PDB accession 4GRV; active-like NTS-R1, PDB accession 4XEE.

  10. Extended Data Figure 10: Model of G-protein-dependent high-affinity agonist binding. (341 KB)

    a, b, As in Fig. 5, nucleotide-free G-protein-stabilized family A GPCRs experience alterations in the extracellular face of the receptor, thus affecting the orthosteric-binding site. In a monoamine receptor such as the β2AR, G-protein binding and GDP loss accompanies the stabilization of a closed, active conformation of the receptor, as in a. b, For family members such as MOPr or NTS-R1, where the peptide hormones/agonists are considerably larger, the influence of the G-protein-mediated changes in the extracellular domain structure result in similar effects on orthosteric ligand dissociation. Rather than closing over the orthosteric site as with monoamine receptors as in a, the extracellular face may contain structures and residues that ‘pinch’ the larger ligands.

Supplementary information

Video

  1. Video 1: Activation of the β2AR (8.01 MB, Download)
    Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). For reference, epinephrine is modeled in the orthosteric binding site. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC). Highlighted are Phe193ECL2 and Tyr3087.35.
  2. Video 2: Comparison of β1AR and β2AR (3.33 MB, Download)
    The closed, active conformation is stabilized by the G protein. Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (cyan, PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). Superimposed on top is a similar morph transitioning between the carazolol-boundββ1-adrenergic receptor (β1AR, lime green, PDB: 2YCW) and isoproterenol-bound (but not G protein- or Nb-bound, PDB: 2Y03). While the β2AR adopts a closed conformation stabilized by agonist and G protein, the β1AR bound only to isoproterenol does not. For reference, epinephrine is modeled in the orthosteric binding site. Note that nanobody Nb80 has been omitted from the animation for simplicity. Highlighted are Phe193ECL2 and Tyr3087.35 on β2AR and the conserved residues Phe201ECL2 and Phe3527.35 on β1AR. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  3. Video 3: Activation of the μ-opioid receptor, MOPr (5.81 MB, Download)
    The ribbon structure of the μ-opioid receptor (MOPr) in its inactive conformation bound to β-funaltrexamine (cyan, PDB: 4DKL) and the MOPr in its active conformation bound to agonist BU72 and Nb39 (orange, PDB: 5C1M). For reference only BU72 is displayed as spheres. Note that Nb39 is not illustrated for simplicity. Morph between the inactive and active conformations was generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  4. Video 4: Activation of the M2 muscarinic receptor, M2R (2.66 MB, Download)
    Top view of the ribbon structure of the M2 muscarinic receptor (M2R) in its inactive conformation bound to antagonist 3-quinuclidinyl benzilate, (QNB, PDB: 3UON) and the M2R in its active conformation bound to agonist iperoxo and nanobody Nb9-8 (PDB: 4MQS). Illustrated are sidechain residues Y1043.33, Y4036.38 and Y4267.39 to highlight the ‘lid-like’ structure over the orthosteric site. Acetylcholine is modeled into the iperoxo binding site and illustrated in stick figure for reference purposes. Note that Nb9-8 is not depicted for simplicity. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  5. Video 5: Activation of the M2R side view (2.55 MB, Download)
    Side view of the ribbon structure of the M2R (above) to highlight the ‘lid-like’ structure over the orthosteric site. Illustrated are side chain residues Y4036.38 and Y4267.39 moving toward Y1043.33 during the formation of the active conformation. Note that TM5 was removed from the rendering so that the tyrosine residues may be easily viewed. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  6. Video 6: Stabilization of the active state of rhodopsin by G protein (5.86 MB, Download)
    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and the photoactivated meta-stable form of rhodopsin bound to the C-terminal fragment of the G protein alpha subunit, transducin (transducin not shown) (PDB:3PAR) (Choe H. W. et al. Crystal structure of metarhodopsin II. Nature471, 651–655 (2011)). Note the pre-existing ‘lid-like’ structure over the orthosteric site formed by the ECL2 and N-terminus. Photoisomerization of 11-cis retinal is illustrated in magenta. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
  7. Video 7: Stabilization of the active state of rhodopsin by arrestin (5.46 MB, Download)
    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and an active mutant of opsin bound to activated arrestin (arrestin not shown) (PDB:4ZWJ) (Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature523, 561-567 (2015)). Note the similarities in conformational changes as with metarhodopsin bound to the C-terminal helix of transducin. Photoisomerization of 11-cis retinal, based on the metarhodopsin structures (Extended data: Movie SM6) has been modeled into the opsin-arrestin structure and is illustrated in magenta. Morphs were generated between inactive and arrestin-bound conformations of rhodopsin using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12.) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

PDF files

  1. Supplementary Information (297 KB)

    This file contains Supplementary Methods, a Supplementary Discussion and additional references.

Comments

  1. Report this comment #68371

    Majid Ali said:

    G-Protein-Coupled Receptors, Stroke Spike, and Cell Membrane Therapeutics

    In a medical setting, hyperventilation feeds upon itself. This is also true of the clinical benefits of slow expirations for controlling hyperventilation, which increase with continued practice. I recognized the former as a surgeon-in-training in England in the mid-1960s. I learned about the latter over twenty years later as a pathologist interested in autonomic regulation with self-regulatory methods. In The Cortical Monkey and Healing (1990, ref. 1), I described Limbic Breathing (a type of slow breathing with prolonged effortless expirations) and its myriad clinical benefits of autonomic regulation, and wondered about some amplification or stability factor that might explain the clinical observations. I suspected that there were some bioenergetic basis for what I observed. The clinical corollary of this was: pathologic factors of disease also fan each other?s fires. When writing Cortical Monkey, I knew that the answers to these questions were clearly beyond my reach.

    DeVree et al provide functional evidence that G-protein coupling to the ?2 adrenergic receptor stabilizes a conformational change in the receptor that restricts the ligand?s access to and egress from the hormone-binding site (ref. 2). In their paradigm, they explain, the active state of the receptor is stabilized by the agonist on one hand and the G protein on the other. The enhancement of agonist affinity arises owing to the positive cooperativity between agonist and G protein--- evolution-designed locks sense and respond to evolutionary keys, so to speak. All physicians interested in self-regulation will be grateful to DeVree and colleagues for this work.

    Autonomic regulation orchestrates regulatory and counter-regulatory influences of the autonomic nervous system, and is very valuable in managing hypertension and avoiding stroke (ref. 3). In one study of 232 patients, following three minutes of Limbic Breathing, average systolic blood pressure levels (in mm Hg) fell by 19, 4, 19, and 12 mm Hg. The corresponding values for reduction in heart rate/minute were 5, 11, 9, and 7.5 respectively (ref. 4). In mild hypertension, systolic blood pressure levels of 140 to 170 usually rise to the 170 to 190 range with incremental stress without specific symptoms experienced by the patient. When subjected to sudden and severe additional stress, systolic blood pressure sometimes rises to 200 to 230 at which level the patients experience a frightening sense of impending catastrophe, which then creates a new sharp spike pushing the pressure to 250 or higher. I coined the term stroke spike to explain to my patients the dangers of such life-threatening surges of blood pressure.

    How does the work of DeVree et al inform clinicians working with autonomic regulation? They found that the effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. This sharedness is expected to cover agonist-receptor dynamics of diverse ligands.

    In Cortical Monkey I included a graph showing blood lactate levels falling by up to 78% with Limbic Breathing for a period of 35 minutes (ref. 5). In 2004, I published evidence for respiratory-to-fermentative shift as the molecular basis of energy deficit in immune-inflammatory disorders (ref. 6). I found that 24-hour urinary excretion of succinate was more common than excretion of other Krebs metabolite. Chouchani et al showed that succinate retention results from reversal of succinate dehydrogenase activity and impaired malate-fumaric shuttle (ref. 7). It is noteworthy in this context that succinate-GPCR91 and a-ketoglutarate-GPCR99 signaling pathways are implicated in the pathogenesis of hypertension (ref. 8).

    In closing, the work of DeVree et al is important to all physicians interested in cell membrane therapeutics, whether prescribing pharmacologic agents which act through GCPRr or natural remedies prescribed for the same purpose.

    References
    1. Ali M. The Cortical Monkey and Healing. Bloomfield, New Jersey. Life Span Books 1991.
    2. DeVree BT, Mahoney JP, Velez-Ruiz, et al. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature 535, 182?186.
    3. Ali M. Oxidative Dysautonomia. In: The Principles and Practice of Integrative Medicine Volume VI: Integrative Cardiology and Chelation Therapies. New York. Canary 21 Press. 2000. 2nd edition 2006.
    4. Ali M. Autonomic Breathing Test (ABT) and its application to 236 patients. Townsend Letter-The Examiner of Alternative Medicine. 2009;315:105-109. October, 2009.
    5. Ali M. The Cortical Monkey and Healing. Bloomfield, New Jersey. Life Span Books 1991, p203.
    6. Ali M. Respiratory-to-Fermentative (RTF) Shift in ATP Production in Chronic Energy Deficit States. Townsend Letter for Doctors and Patients. 2004. 253: 64-65 (2004).
    7. Chouchani ET. Pel VR, Gaude E, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature;515:431.
    8. He W, Milao FJ-P, Lin DC-H, et al. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature. 2004;429:143-45.

Subscribe to comments

Additional data