G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human b2 adrenergic receptor (b2AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive b2AR structure reveals subtle changes in the binding

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Structure of a nanobody-stabilized active state of the β2 adrenoceptor

In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.

https://pharmrev.aspetjournals.org/content/pharmrev/63/1/1.full.pdf

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or “accessory” proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies.

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G Protein-Coupled Receptor Allosterism and Complexing

Adenosine receptors are G protein-coupled receptors that are found in the heart and the brain, and adenosine is the endogenous ligand for this class of transmembrane receptor. Lebon et al. present two X-ray crystal structures of a thermostabilized human adenosine A2A receptor bound to its endogenous agonist adenosine and the synthetic agonist NECA. Comparison of the agonist-bound structures of A2A receptor with the agonist-bound structures of β-adrenoceptors suggests that the contraction of the ligand binding pocket caused by the inward motion of several helices may be a common feature in the activation of all G protein-coupled receptors. Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine1 or noradrenaline2, respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism3,4. Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors5,6,7. Here we present two crystal structures of the thermostabilized human adenosine A2A receptor (A2AR-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 2777.42 and His 2787.43; superscripts refer to Ballesteros–Weinstein numbering9) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A2AR with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

/pdf/agonist-bound-adenosine-a2a-receptor-structures-reveal-ry1pih5f8m.pdf

Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 A resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

/pdf/structure-and-function-of-an-irreversible-agonist-b2-3mkeqoh30a.pdf

Structure and function of an irreversible agonist-β2 adrenoceptor complex

The G protein coupled receptors (GPC-Rs) comprise a large superfamily of genes encoding numerous receptors which all show common structural features, e.g., seven putative membrane spanning domains. Their biological functions are extremely diverse, ranging from vision and olfaction to neuronal and endocrine signaling. The GPC-Rs couple via multiple G proteins to a growing number of recognized second messenger pathway, e.g., cAMP and phosphatidyl inositol turnover. This review summarizes our current knowledge of the molecular mechanisms by which the GPC-Rs activate second messenger systems, and it addresses their regulation and structure.

Structure and Function of G Protein Coupled Receptors

Although vasopressin V 1B receptor (V 1B -R) mRNA was detected in the kidney, the precise renal 27 localization, pharmacological and physiological properties of this receptor remain unknown. Using the 28 selective V 1B agonist d[Leu 4 , Lys 8 ]VP, either fluorescent or radioactive, we showed that V 1B -R is 29 mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. 30 Protein and mRNA expression of V 1B -R were very low compared to V 2 receptor (V 2 -R). On 31 microdissected IMCD, d[Leu 4 , Lys 8 ]VP had no effect on cAMP production but induced a dose- 32 dependent and saturable [Ca 2+ ] i increase mobilization with an EC 50 in the nanomolar range. This effect 33 involved both intracellular calcium mobilization and extracellular calcium influx. The selective V 1B 34 antagonist SSR149415 strongly reduced the ability of vasopressin to increase [Ca 2+ ] i but also cAMP 35 suggesting a cooperation between V 1B -R and V 2 -R in IMCD cells expressing both receptors. This 36 cooperation arises from a crosstalk between second messenger cascade involving protein kinase C 37 rather than receptor heterodimerization as supported by potentiation of the AVP-stimulated cAMP 38 production in HEK293 cells co-expressing the two receptor isoforms and negative results obtained by 39 bioluminescence resonance energy transfer experiments. In vivo , only acute administration of high 40 doses of V 1B agonist triggered significant diuretic effects by contrast with injection of selective V 2 41 agonist. This study brings new data on the localization and signaling pathways of V 1B -R in the kidney, 42 highlights a crosstalk between V 1B -R and V 2 -R in IMCD and suggests that V 1B -R may counterbalance 43 in some pathophysiological conditions the antidiuretic effect triggered by V 2 -R activation. Gapdh mRNA used as internal control. After an initial denaturation step of 10 min at 95°C, the thermal cycling conditions were 40 cycles at 95°C for 15 s and 60°C for 1 min. Each sample was tested in triplicate. Data were normalized to the expression levels of GAPDH mRNA. pre-coated poly-ornithine the cells/well and transfected V 1B -R and/or V 2 -R plasmids using Lipofectamine 2000 according the manufacturer. DNA constructs were generated by PCR amplification using the full-length human wild-type V 1B -R (25) and V 2 -R (kindly as previously described. Forty-eight hours after transfection, cells were pretreated with PMA (15 min, 1 µmol/L) and/or Gö6976 (30 min, 100 nmol/L), then treated for 30 min at 37°C with or without AVP in the presence of phosphodiesterase inhibitor (RO201724 0.1mmol/L). The produced was detected using the HTRF Gs Bioassay (Dynamic 2 kit, CisBio). Plates were read on a PHERAstar FS (BMG The specific FRET signal was calculated using the following equation: ΔF%= 100 x (Rpos-Rneg)/(Rneg), with Rpos being the fluorescence ratio (665/620nm) calculated in wells incubated with both donor-labeled antibody and acceptor d2, and Rneg being the same ratio for the negative control incubated only with the donor fluorophore-labeled antibody. The FRET signal (ΔF%), inversely proportional to the quantity of cAMP, was then transformed in cAMP concentration using a calibration curve prepared on the same plate. Specific binding calculated in each experimental are expressed as percent and are the mean ± SE of 3 experiments each in

Characterization of a functional V 1B vasopressin receptor in the male rat kidney, evidence for crosstalk between V 1B and V 2 receptor signaling pathways

Maithe Corbani

Papers

Structure and Function of G Protein Coupled Receptors

Characterization of a functional V 1B vasopressin receptor in the male rat kidney, evidence for crosstalk between V 1B and V 2 receptor signaling pathways