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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 mechanism 1 . Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric G i . 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 G i 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. Cryo-electron microscopy structures show that metabotropic glutamate receptor 2 forms a dimer to which only one G protein is coupled, revealing the basis for asymmetric signal transduction.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca 2+ , is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders 1 . CaSR is a family C G-protein-coupled receptor 2 that functions as an obligate homodimer, with each protomer composed of a Ca 2+ -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 Ca 2+ 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. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor 1 (GPCR) that has a central role in regulating systemic calcium homeostasis 2 , 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 G i versus G q proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both G i and G q 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 G i and G q 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. Cryo-electron microscopy structures of the human calcium-sensing receptor in complex with G i and G q proteins reveal how this receptor activates distinct G protein subtypes and how its function is modulated by a variety of ligands.

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 β 2 adrenergic receptor (β 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 β 2 AR and nucleotide-free Gs heterotrimer. The principal interactions between the β 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 β 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. X-ray structure of a GPCR complex G-protein-coupled receptors (GPCRs) mediate the majority of a cell's responses to hormones and neurotransmitters, and to the senses of sight, olfaction and taste. This makes GPCRs potentially the most important group of drug targets in the human body. GPCRs are deeply embedded in the cell membrane, crossing it seven times, so structure determination for these complexes is particularly challenging — as recounted in a recent News Feature (see http://go.nature.com/ftqnx4 ). The eagerly-awaited X-ray crystal structure of a GPCR transmembrane signalling complex has now been determined by Brian Kobilka's group. The structure presented is of an agonist-occupied monomer of the β 2 adrenergic receptor in complex with G s , the stimulatory G protein for adenylyl cyclase. An accompanying paper reports the use of peptide amide hydrogen-deuterium exchange mass spectrometry to probe the protein dynamics of this signalling complex.

Stimulation of the metabotropic GABA B receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology 1 . GABA B 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 proteins 2 , 3 . However, GABA B is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits 4 , 5 . Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA B receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABA B 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 GABA B 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 GABA B dimers, and have important implications for rational drug design that targets these receptors. Cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA B receptors, combined with cellular signalling assays, shed light on the mechanisms that underpin signal transduction mediated by these 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. The activation mechanism of metabotropic glutamate receptor subtype 5, a member of the family C G-protein-coupled receptors, is characterized by a combination of cryo-electron microscopy, crystallography and signalling studies.

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. The crystal structure of the mouse μ-opioid receptor bound to an antagonist is described, with possible implications for the future development of analgesics. Where opiates hit home Four papers in this issue of Nature present the long-awaited high-resolution crystal structures of the four known opioid receptors in ligand-bound conformations. These G-protein-coupled receptors are the targets of a broad range of drugs, including painkillers, antidepressants, anti-anxiety agents and anti-addiction medications. Brian Kobilka’s group reports the crystal structure of the µ-opioid receptor bound to a morphinan antagonist and the δ-opioid receptor bound to naltrindole. Raymond Stevens’ group reports on the κ-opioid receptor bound to the selective antagonist JDTic, and the nociceptin/orphanin FQ receptor bound to a peptide mimetic. In an associated News and Views, Marta Filizola and Lakshmi Devi discuss the implications of these landmark papers for research on the mechanisms underlying receptor function and drug development.

The µ-opioid receptor (µOR) is a well-established target for analgesia 1 , yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl 2 , a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to ‘steer’ hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo. A newly discovered negative allosteric modulator of the µ-opioid receptor works together with naloxone to potently block opioid agonist signalling with reduced adverse effects.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound G s protein (G s ·GDP; hereafter G s GDP ) upon coupling to the β 2 -adrenergic receptor (β 2 AR) in atomic detail. The binding of G s GDP to the β 2 AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the G s occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this β 2 AR–G s GDP intermediate, G s undergoes an in-plane rotation along the receptor axis to approach the β 2 AR–G s empty state. The simulations shed light on how the structural elements at the receptor–G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity. Using molecular dynamics simulations and functional assays, authors track the structural changes in heterotrimeric G proteins in response to receptor coupling that lead to the ejection of GDP, the rate-limiting step during G-protein activation.

Most G protein-coupled receptors (GPCRs) recruit β-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 β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-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 β-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 β-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 β-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.