Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3',5'-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs , Gq/11 and G12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1 , Gi2 , Gi3 , GoA , GoB and Gz ). The Emax and activation rates of Gi1, Gi2 , Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

G-protein activation by a metabotropic glutamate receptor.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

Asymmetric activation of the calcium-sensing receptor homodimer.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor.

Dissecting the roles of GRK2 and GRK3 in µ-opioid receptor internalization and ß-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells.

Most G protein-coupled receptors (GPCRs) recruit ß-arrestins and internalize upon agonist stimulation. For the µ-opioid receptor (µ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for µ-OR recruitment of ß-arrestin and internalization. However, the contribution of GRK2 and GRK3 to ß-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in ß-arrestin2 recruitment and µ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced µ-OR internalization and ß-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of ß-arrestin2 recruitment to the plasma membrane upon µ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs.

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11 subfamily consists of Gq, G11, G14, and G16 proteins, of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, Gs, Gi/o, and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o, and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359-sensitive Gαs mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.

Structures of metabotropic GABAB receptor.

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors.

Structural Insights into the Process of GPCR-G Protein Complex Formation.

The crystal structure of the ß2-adrenergic receptor (ß2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (ß2AR-Gsempty). Unfortunately, the ß2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the ß2AR and GDP-bound Gs protein (ß2AR-GsGDP) that may represent an intermediate on the way to the formation of ß2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the ß2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the ß2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.

Author Correction: Structural insights into the activation of metabotropic glutamate receptors.

The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.

Structural insights into the activation of metabotropic glutamate receptors.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

mGluR5: exploration of orthosteric and allosteric ligand binding pockets and their applications to drug discovery.

Since its discovery in 1992, mGluR5 has attracted significant attention and been linked to several neurological and psychiatric diseases. Ligand development was initially focused on the orthosteric binding pocket, but lack of subtype selective ligands changed the focus to the transmembrane allosteric binding pocket. This strategy has resulted in several drug candidates in clinical testing. In the present article we explore the orthosteric and allosteric binding pockets in terms of structure and ligand recognition across the mGluR subtypes and groups, and discuss the clinical potential of ligands targeting these pockets. We have performed binding mode analyses of non- and group-selective orthosteric ligands based on molecular docking in mGluR crystal structures and models. For the analysis of the allosteric binding pocket we have combined data from all mGluR5-mutagenesis studies, collectively reporting five negative allosteric modulators and 47 unique mutations, and compared it to the closest related homolog, mGluR1.

Pharmacological characterization and modeling of the binding sites of novel 1,3-bis(pyridinylethynyl)benzenes as metabotropic glutamate receptor 5-selective negative allosteric modulators.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 µM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.

Crystal structure of the µ-opioid receptor bound to a morphinan antagonist.

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

Structure-activity relationships for negative allosteric mGluR5 modulators.

A series of compounds based on the mGluR5-selective ligand 2-methyl-6-(phenylethynyl)pyridine (MPEP) were designed and synthesized. The compounds were found to be either structural analogues of MPEP, substituted monomers, or dimeric analogues. All compounds retained mGluR5 selectivity with only weak or no activity at other mGluRs or iGluRs. The substituted analogue, 1,3-bis(pyridin-2-ylethynyl)benzene (19), is a potent negative modulator at mGluR5, whereas all other compounds lost potency relative to MPEP and showed that activity is highly dependent on the position of the nitrogen atom in the pyridine moieties. A homology modeling and ligand docking study was used to understand the binding mode and the observed selectivity of compound 19.

Crystal structure of the ß2 adrenergic receptor-Gs protein complex.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ß(2) adrenergic receptor (ß(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ß(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ß(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ß(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

Exploration of SAR features by modifications of thiazoleacetic acids as CRTH2 antagonists.

The SAR features have been further explored for (2-benzhydryl-4-phenyl-thiazol-5-yl)acetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. The introduction of a nitrogen or a methyl substituent in the benzhydrylic position offer two alternative drugable scaffolds attractive for unsymmetrically substituted derivatives. An imidazole analogue lacks activity due to formation of a favored coplanar intramolecular hydrogen bond. The pyrimidine derivative 18 represents a potent and selective compound that will be subject to continued investigations.

Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 2.

Structure-activity relationships have been established by exploring the eastern and western side of 5-thiazolyleacetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Benzhydryl motifs in the 2-position of the thiazole was found to be most advantageous. The 4-thiazole position should either carry 3- or 4-fluorophenyl rings or a 4-pyridyl suitably substituted in the flanking 2-position. Several compounds with single digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained. The compound series display a good PK profile and selectivity over a large number of other targets.