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. 2009 Jun 9;106(23):9501-6.
doi: 10.1073/pnas.0811437106. Epub 2009 May 22.

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

Xiao Jie Yao  1 , Gisselle Vélez Ruiz, Matthew R Whorton, Søren G F Rasmussen, Brian T DeVree, Xavier Deupi, Roger K Sunahara, Brian Kobilka
Affiliations

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

Xiao Jie Yao et al. Proc Natl Acad Sci U S A. .

Abstract

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Site-specific labeling of purified β2AR with monobromobimane. (A) Sequence and secondary structure of the human β2AR showing sites where reactive cysteines were mutated to the indicated amino acid (black circles with white letters). C265 is shown in red. The remaining cysteines (gray circles) are not reactive toward monobromobimane. (B) The structure of bimane covalently bound to C265 after reaction with monobromobimane. (C) In the inactive structure of the β2AR, bimane bound to C265 is predicted to occupy a cavity between TM3, TM5, and TM6. When TM6 adopts an active conformation (because of agonist binding or due to constitutive activity), bimane is displaced (black arrow) out of this cavity to a more polar environment, which is detected as a change in fluorescence intensity and λMAX. (D) A model of the active state of the β2AR in complex with the carboxyl terminal peptide of GαS, based on the crystal structure of opsin in complex with a transducin peptide. The amino acids at the positions marked by solid spheres are predicted to form interactions with GαS. The residues of the β2AR are numbered according to their position in the sequence followed by the Ballesteros general number (49) in superscript. In this numbering scheme, the most conserved residue within each helix is designated x.50, where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position. The model of the active conformation of β2AR shown in Fig. 1D was built by homology modeling using the β2AR (2RH1) and opsin (3CAP) structures as templates. The Gαs fragment bound to the active-like β2AR was modeled by threading the 11 C-terminal residues of Gαs on the structure of the synthetic peptide derived from the carboxy terminus of Gαt bound to opsin. The surface of the cytoplasmic side of the receptor shown in Fig. 1C was calculated with CastP (50). Homology models and figures were prepared with PyMOL (DeLano WL (2002) The PyMOL Molecular Graphics System. Available at http://www.pymol.org).
Fig. 2.
Fig. 2.
Reconstitution of purified bimane-labeled β2AR (mB-β2AR) and Gs into rHDL particles. (A) Bimane emission spectra of reconstituted mB-β2AR in the absence (black spectrum) and presence of increasing concentrations of the agonist ISO (red spectra). Agonist-induced conformational changes lead to a decrease in fluorescence intensity of bimane and a shift in the λMAX. (B and C) Bimane emission spectra of mB-β2AR reconstituted with Gs in the absence of agonist or nucleotides (black spectrum) and in the presence of increasing concentrations of GTPγS (B) and GDP (C) (red spectra). All spectra were normalized to the unliganded, uncoupled state of mB-β2AR. For experiments in A, this state was the value for the sample without ligand. For experiments in B and C, this state was the value obtained following the addition of 10 μM GTPγS. Each spectrum in A–C represents the average of 3 independent experiments. (D) ISO inhibition of [3H]DHAP binding to mB-β2AR in rHDL in the presence (red) and absence (black) of Gs. Inset shows the effect of 10 μM GTPγS on ISO bind affinity to mB-β2AR reconstituted with Gs. These data were fit by using Prism 5.0 (Graphpad). (E) Time-scan of fluorescence monitored at 450 nm. The effect of 200 nM GTP, GTPγS, and GDP on mB-β2AR-Gs are compared. GTP induces dissociation of mB-β2AR and Gs followed by reformation of the complex after GTP hydrolysis.
Fig. 3.
Fig. 3.
The effect of ligands having different efficacies on the stability of mB-β2AR-Gs. Emission scans were performed on mB-β2AR-Gs complex obtained from a single reconstitution. For each treatment (apyrase, ISO, alprenolol, ICI, or GTPγS), a baseline scan was performed on mB-β2AR-Gs alone (black spectrum), and subsequent scans were normalized to this baseline scan. (A) The gray spectrum was taken 10 min after the addition of 10 μM GTPγS to mB-β2AR-Gs. This treatment uncouples Gs from mB-β2AR. The blue spectrum shows the effect of a 40-min incubation of mB-β2AR-Gs with apyrase to remove residual GDP. (A–C) The effect of ligands was determined after 40-min pretreatment with apyrase (blue) followed by a 60-min incubation with the ligand. A: ISO, green; B: alprenolol, purple; C: ICI, red. This experiment is representative of 3 independent experiments.
Fig. 4.
Fig. 4.
The effect of ligand efficacy on Gs-induced changes in mB-β2AR fluorescence. Initial emission scans (black spectra) of mB-β2AR were obtained, then scans were repeated after a 15-min incubation with the indicated ligands (blue spectra). A concentrated solution of Gs was added (1:100 dilution of an 8 mg/mL solution), and emission scans were repeated after 8 min (red spectra). Preliminary studies showed that the effect of Gs was complete at 6 min. Ligands: A, no ligand; B, isoproterenol (Iso); C, ICI; D, alprenolol (Alp); E, carazolol (Cz). These scans are representative of 3 independent experiments.
Fig. 5.
Fig. 5.
Conceptual model depicting the dynamic behavior of β2ARs. In the absence of a ligand, the β2AR exists in an ensemble of basal states in dynamic equilibrium (blue background). Agonists and inverse agonists bind to and stabilize distinct substates. The nucleotide free form of the G protein Gs can also bind to and stabilize an active state of the β2AR (green background). The different equilibrium processes between the receptor and its ligands and the receptor and the G protein Gs are displayed (A–H) and are described in detail in the text. Note that for each of these equilibria, the relative size of the arrows indicates the displacement of the reaction.
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