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G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different [alpha]-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G.sub.s to four different GPCRs have been elucidated.sup.1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT.sub.1B receptor (5-HT.sub.1BR) bound to the agonist donitriptan and coupled to an engineered G.sub.o heterotrimer. In this complex, 5-HT.sub.1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the [beta].sub.2-adrenoceptor ([beta].sub.2AR).sup.3 or the adenosine A.sub.2A receptor (A.sub.2AR).sup.1 in complex with G.sub.s. In contrast to the complexes with G.sub.s, the gap between the receptor and the G[beta]-subunit in the G.sub.o-5-HT.sub.1BR complex precludes molecular contacts, and the interface between the G[alpha]-subunit of G.sub.o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G.sub.o [alpha]-subunit. The molecular variations between the interfaces of G.sub.o and G.sub.s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling. The high-resolution structure of the serotonin 5-HT.sub.1B receptor in complex with the agonist donitriptan and a G.sub.o heterotrimer highlights features that may underlie the specificity of receptor-G-protein coupling and kinetics of signalling., Author(s): Javier García-Nafría [sup.1] , Rony Nehmé [sup.1] , Patricia C. Edwards [sup.1] , Christopher G. Tate [sup.1] Author Affiliations: (1) MRC Laboratory of Molecular Biology, Cambridge, UK Main Heterotrimeric [...]

Author(s): A. J. Venkatakrishnan (corresponding author) [1, 2, 3, 4]; Xavier Deupi [5]; Guillaume Lebon [6, 7, 8]; Franziska M. Heydenreich [5, 9, 10]; Tilman Flock [1]; Tamara Miljus [5, [...]

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary (1,2). Here we present the structure of the adenosine [A.sub.2A] receptor ([A.sub.2A]R) bound to an engineered G protein, mini-[G.sub.s], at 3.4 Å resolution. Mini-[G.sub.s] binds to [A.sub.2A]R through an extensive interface (1,048 [Å.sup.2]) that is similar, but not identical, to the interface between [G.sub.s] and the [β.sub.2]-adrenergic receptor (3). The transition of the receptor from an agonist-bound active-intermediate state (4,5) to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains [Arg.sup.3.50], [Tyr.sup.5.58] and [Tyr.sup.7.53]. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The [A.sub.2A]R-mini-[G.sub.s] structure highlights both the diversity and similarity in G-protein coupling to GPCRs (6) and hints at the potential complexity of the molecular basis for G-protein specificity., Structures of [A.sub.2A]R bound to either inverse agonists (7-9) or agonists (4,5,10) have elucidated the molecular determinants of subtype specificity and ligand efficacy (11). However, the mechanism of activation of [...]

G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ~800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism., G proteins bind guanine nucleotides and act as molecular switches in a number of signalling pathways by interconverting between a GDP-bound inactive and a GTP-bound active state (1,2). They consist [...]

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine., Signal transduction is a fundamental biological process that is required to maintain cellular homeostasis and to ensure coordinated cellular activity in all organisms. Membrane proteins at the cell surface serve [...]

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to [NTS.sub.8-13], the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity., Neurotensin (NTS) is a short peptide that is found in the nervous system and in peripheral tissues (1). NTS shows a wide range of biological activities and has important roles [...]

Adenosine A.sub.2A receptor structure 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 A.sub.2A receptor bound to its endogenous agonist adenosine and the synthetic agonist NECA. Comparison of the agonist-bound structures of A.sub.2A receptor with the agonist-bound structures of [beta]-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 [beta]-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine.sup.1 or noradrenaline.sup.2, 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 mechanism.sup.3,4. Recent structures of [beta]-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 receptors.sup.5,6,7. Here we present two crystal structures of the thermostabilized human adenosine A.sub.2A receptor (A.sub.2AR-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 277.sup.7.42 and His 278.sup.7.43; superscripts refer to Ballesteros-Weinstein numbering.sup.9) 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 A.sub.2AR with the agonist-bound structures of [beta]-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., Author(s): Guillaume Lebon [sup.1] , Tony Warne [sup.1] , Patricia C. Edwards [sup.1] , Kirstie Bennett [sup.2] , Christopher J. Langmead [sup.2] , Andrew G. W. Leslie [sup.1] , Christopher [...]

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline (1, 2). Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) [β.sub.1]-adrenergic receptor ([β.sub.1]AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 ([Ser.sup.5.42] and [Ser.sup.5.46]), but partial agonists only interact with [Ser.sup.5.42] (superscripts refer to Ballesteros-Weinstein numbering (3)). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies., Determining how agonists and antagonists bind to the b receptors has been the goal of research for more than 20 years (4-11). Although the structures of the homologous [β.sup.1] and [...]

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7Å resolution crystal structure of a [beta.sub.1]-adrenergic receptor [...]

Author(s): Byron Carpenter; Rony Nehm; Tony Warne; Andrew G. W. Leslie; Christopher G. Tate Nature 536, 104107 (2016); doi:10.1038/nature18966 In this Letter, author B.C. (http://byronc@mrc-lmb.cam.ac.uk ) should have also been [...]

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1–Ste4–Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4. A cryo-electron microscopy structure of the yeast pheromone receptor Ste2, a class D G-protein-coupled receptor, in its active state reveals that Ste2 is a homodimer that couples to two G proteins. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated [1]- [4], but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β2-adrenoceptor (β2AR) [3] or the adenosine A2A receptor (A2AR) [1] in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gβ-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling. The high-resolution structure of the serotonin 5-HT1B receptor in complex with the agonist donitriptan and a Go heterotrimer highlights features that may underlie the specificity of receptor-G-protein coupling and kinetics of signalling. [ABSTRACT FROM AUTHOR]

Adenosine receptors and ?-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, 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 mechanism. Recent structures of ?-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 receptors. 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 numbering) 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 ?-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. [ABSTRACT FROM AUTHOR]

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46), but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. [ABSTRACT FROM AUTHOR]

The β 1 -adrenoceptor (β 1 AR) is a G-protein-coupled receptor (GPCR) that couples 1 to the heterotrimeric G protein G s . G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of β-arrestin 1 (βarr1, also known as arrestin 2), which displaces G s and induces signalling through the MAP kinase pathway 2 . The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism 3 -is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects 4 . To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the β 1 AR-βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol 5 , and the crystal structure of formoterol-bound β 1 AR coupled to the G-protein-mimetic nanobody 6 Nb80. βarr1 couples to β 1 AR in a manner distinct to that 7 of G s coupling to β 2 AR-the finger loop of βarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of G s . The conformation of the finger loop in βarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin 8 . β 1 AR coupled to βarr1 shows considerable differences in structure compared with β 1 AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of β 1 AR, and find that formoterol has a lower affinity for the β 1 AR-βarr1 complex than for the β 1 AR-G s complex. The structural differences between these complexes of β 1 AR provide a foundation for the design of small molecules that could bias signalling in the β-adrenoceptors.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1 . Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P 2 binding. Endogenous lipids were also observed bound directly to the trimeric Gα s βγ protein complex of the adenosine A 2A receptor (A 2A R) in the gas phase. Using engineered Gα subunits (mini-Gα s, mini-Gα i and mini-Gα 12 ) 2 , we demonstrate that the complex of mini-Gα s with the β 1 adrenergic receptor (β 1 AR) is stabilized by the binding of two PtdIns(4,5)P 2 molecules. By contrast, PtdIns(4,5)P 2 does not stabilize coupling between β 1 AR and other Gα subunits (mini-Gα i or mini-Gα 12 ) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P 2 . Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gα i βγ complex in the presence of PtdIns(4,5)P 2 provide further evidence for a specific effect of PtdIns(4,5)P 2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P 2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.