Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor - PubMed (original) (raw)

. 2010 Jan 7;463(7277):108-12.

doi: 10.1038/nature08650.

Yaozhong Zou, Søren G F Rasmussen, Corey W Liu, Rie Nygaard, Daniel M Rosenbaum, Juan José Fung, Hee-Jung Choi, Foon Sun Thian, Tong Sun Kobilka, Joseph D Puglisi, William I Weis, Leonardo Pardo, R Scott Prosser, Luciano Mueller, Brian K Kobilka

Affiliations

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

Michael P Bokoch et al. Nature. 2010.

Abstract

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

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Figures

Figure 1

Figure 1. Extracellular domains of carazolol-bound β2AR (PDB: 2RH1)

a, The extracellular surface (ECS) of β2AR showing extracellular loop 2 (ECL2, cyan, Met171-Ala198), extracellular loop 3 (ECL3, dark blue, His296-Glu306), Lys3057.32 (magenta), Asp192 (yellow), and inverse agonist carazolol (green). ECL1 (Met96-Phe108) is part of the ECS but is not colored. b, Intramolecular and ligand binding interactions. Spheres indicate the alpha carbons of residues in direct contact with carazolol (at least one atom within 4 Å distance). Disulfide bonds are shown as yellow sticks. Other colors are the same as in a. Transmembrane helices 1 and 2 removed for clarity. D192 and K305 form the only lysine salt bridge observed in the crystal structure. The solvent accessibility of D192 and K305 was calculated with the NACCESS program (Hubbard & Thornton). The relative accessibilities of D192 and K305 are 35% and 75%, respectively, compared to the accessibility of that residue type in an extended Ala-x-Ala tripeptide.

Figure 2

Figure 2. Dimethyllysine NMR spectroscopy of [13C]methyl-β2AR and assignment of Lys305

a, Dimethylamine region of STD-filtered HC-HMQC spectrum of carazolol-bound β2AR365, containing 14 Lys and amino terminal FLAG-TEV sequence (Supplementary Fig. 2). b, Spectrum of β2AR365 with seven cytoplasmic Lys to Arg mutations (β2AR365 Δ7Lys) and the amino terminus removed by TEV proteolysis. c, Spectrum of β2AR365 Δ7Lys plus the Lys305Arg mutation and the amino terminus removed by TEV proteolysis.

Figure 3

Figure 3. Effect of inverse agonist and antagonist on the [13C]dimethyl-Lys305 NMR resonances

HSQC spectra of a, unliganded β2AR (≈60 μM), b, β2AR bound to inverse agonist carazolol and c, β2AR bound to neutral antagonist alprenolol.

Figure 4

Figure 4. Activation of β2AR by formoterol

STD-filtered HMQC spectra of a, unliganded β2AR (≈60 μM), b, the same sample bound to a saturating concentration (320 μM) of agonist (R,R)-formoterol, and c, the same sample after exchanging formoterol for inverse agonist carazolol by dialysis. d, Model of β2AR activation by formoterol (see Supplementary Fig. 16). Colored helices, loops, and side chains represent the carazolol-bound crystal structure (PDB: 2RH1). Gray helices and white side chains indicate the active state model. Green sticks indicate (R,R)-formoterol and yellow indicates ECL2. e, Overlay of spectra corresponding to dashed regions shown in panels a-c. The spectrum of unliganded β2AR from panel a is shown in black, agonist-bound β2AR from panel b in green, and inverse agonist-bound β2AR from panel c in red.

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References

    1. Rosenbaum DM, et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science. 2007;318:1266–73. - PubMed
    1. Cherezov V, et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007;318:1258–65. - PMC - PubMed
    1. Rasmussen SG, et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007;450:383–7. - PubMed
    1. Jaakola VP, et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science. 2008;322:1211–7. - PMC - PubMed
    1. Warne T, et al. Structure of a beta1-adrenergic G-protein-coupled receptor. Nature. 2008;454:486–91. - PMC - PubMed

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