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Phosphorylation of myofilament proteins critically regulates beat-to-beat cardiac contraction and is typically altered in heart failure (HF). β-Adrenergic activation induces phosphorylation in numerous substrates at the myofilament. Nevertheless, how cardiac β-adrenoceptors (βARs) signal to the myofilament in healthy and diseased hearts remains poorly understood. The aim of this study was to uncover the spatiotemporal regulation of local βAR signaling at the myofilament and thus identify a potential therapeutic target for HF. Phosphoproteomic analysis of substrate phosphorylation induced by different βAR ligands in mouse hearts was performed. Genetically encoded biosensors were used to characterize cyclic adenosine and guanosine monophosphate signaling and the impacts on excitation-contraction coupling induced by β1AR ligands at both the cardiomyocyte and whole-heart levels. Myofilament signaling circuitry was identified, including protein kinase G1 (PKG1)-dependent phosphorylation of myosin light chain kinase, myosin phosphatase target subunit 1, and myosin light chain at the myofilaments. The increased phosphorylation of myosin light chain enhances cardiac contractility, with a minimal increase in calcium (Ca2+) cycling. This myofilament signaling paradigm is promoted by carvedilol-induced β1AR-nitric oxide synthetase 3 (NOS3)-dependent cyclic guanosine monophosphate signaling, drawing a parallel to the β1AR-cyclic adenosine monophosphate-protein kinase A pathway. In patients with HF and a mouse HF model of myocardial infarction, increasing expression and association of NOS3 with β1AR were observed. Stimulating β1AR-NOS3-PKG1 signaling increased cardiac contraction in the mouse HF model. This research has characterized myofilament β1AR-PKG1-dependent signaling circuitry to increase phosphorylation of myosin light chain and enhance cardiac contractility, with a minimal increase in Ca2+ cycling. The present findings raise the possibility of targeting this myofilament signaling circuitry for treatment of patients with HF.

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 [...]

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 [...]

There are [approximately equal to]350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermo-stable mutants of a GPCR, the [beta]1-adrenergic receptor. The most stable mutant receptor, [beta]AR-m23, contained six point mutations that led to an apparent [T.sub.m] 21[degrees]C higher than the native protein, and, in the presence of bound antagonist, [beta]AR-m23 was as stable as bovine rhodopsin. In addition, [beta]AR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand. G protein-coupled receptor | membrane protein | stabilization