Showing papers by "Michel Bouvier published in 2006"

Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes

Abstract: Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor–G protein complexes. Using the data obtained for the α2AAR–Gαi1β1γ2 complex and the available crystal structures of Gαi1β1γ2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor–G protein complex, leading the Gα-Gβγ interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the Gα subunit, thus reflecting the initial activation event.

Distinct signaling profiles of beta1 and beta2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy.

Abstract: Drug efficacy is typically considered an intrinsic property of a ligand/receptor couple. However, recent observations suggest that efficacy may also be influenced by the signaling effectors engaged by a unique receptor. To directly and systematically test this possibility, we assessed the ability of a panel of beta-adrenergic ligands to modulate the activity of two effector systems, the adenylyl cyclase (AC) and the mitogen-activated protein kinase (MAPK), via beta(1) and beta(2) adrenergic receptors. Although some compounds displayed similar efficacies toward the two pathways, others showed complex efficacy profiles. For example, compounds that are inverse agonists for the AC activity were found to be either agonists, neutral antagonists, or inverse agonists for the MAPK pathway. Likewise, agonists for the AC were either agonists or neutral antagonists for MAPK. Given this complexity, we propose a Cartesian representation of the efficacies that takes into account the activities of the different effectors that can be engaged by a given receptor. In addition, compounds considered as nonselective for beta(1) and beta(2) adrenergic receptors, based on their binding affinities, showed distinct relative efficacy profiles toward AC and MAPK, adding a new dimension to the concept of ligand selectivity. Taken together, the results suggest that binding of different ligands promote distinct conformational changes leading to specific signaling outcomes. Our data therefore clearly illustrate that efficacy is a pluridimensional parameter that is not an intrinsic characteristic of a ligand/receptor couple. This should have important implications for the future design of screening assays used in drug discovery campaigns.

Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.

Abstract: Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.

Monitoring protein-protein interactions in living cells by bioluminescence resonance energy transfer (BRET).

Abstract: Bioluminescence resonance energy transfer (BRET) allows monitoring of protein-protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such as Renilla luciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 A, making the technique ideal for monitoring protein-protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist-promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of beta-arrestin2 to agonist-activated G-protein-coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.

Simultaneous Activation of the δ Opioid Receptor (δOR)/Sensory Neuron-Specific Receptor-4 (SNSR-4) Hetero-Oligomer by the Mixed Bivalent Agonist Bovine Adrenal Medulla Peptide 22 Activates SNSR-4 but Inhibits δOR Signaling

Abstract: Hetero-oligomerization among G protein-coupled receptors has been proposed to contribute to signal integration. Because sensory neuron-specific receptors (SNSRs) and the opioid receptors (OR) share a common ligand, the bovine adrenal medulla peptide (BAM) 22, and have opposite effects on pain modulation, we investigated the possible consequences of deltaOR/SNSR-4 hetero-oligomerization on the signaling properties of both receptor subtypes. Bioluminescence resonance energy transfer revealed that the human deltaOR has similar propensity to homo-oligomerize and to form hetero-oligomers with human SNSR-4 when coexpressed in human embryonic kidney 293 cells. The hetero-oligomerization leads to a receptor form displaying unique functional properties. Individual activation of either deltaOR or SNSR-4 in cells coexpressing the two receptors led to the modulation of their respective signaling pathways; inhibition of adenylyl cyclase and activation of phospholipase C, respectively. In contrast, the deltaOR/SNSR-4 bivalent agonist BAM22, which could activate each receptor expressed individually, fully activated the SNSR-4-dependent phospholipase C but did not promote deltaOR-mediated inhibition of adenylyl cyclase in deltaOR/SNSR-4-coexpressing cells. Likewise, concomitant activation of the deltaOR/SNSR-4 hetero-oligomer by selective deltaOR and SNSR-4 agonists promoted SNSR-4 but not deltaOR signaling, revealing an agonist-dependent dominant-negative effect of SNSR-4 on deltaOR signaling. Furthermore, the deltaOR selective antagonist naltrexone trans-inhibited the SNSR-4-promoted phospholipase C activation mediated by BAM22 but not by the SNSR-4-selective agonists, suggesting a bivalent binding mode of BAM22 to the deltaOR/SNSR-4 hetero-oligomer. The observation that BAM22 inhibited the Leu-enkephalin-promoted cAMP inhibition in rat dorsal root ganglia neurons supports the potential physiological implication of such regulatory mechanism.

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy.

Abstract: The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TATPep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.

An aplysia dopamine1‐like receptor: molecular and functional characterization

Abstract: In Aplysia, the neurotransmitter dopamine is involved in the regulation of various physiological processes and motor functions, like feeding behaviour, and in the siphon-gill withdrawal reflex. In this paper, we report the characterization of the first Aplysia D1-like dopamine receptor (Apdop1) mainly expressed in the CNS, heart and buccal mass. Following expression of the Apdop1 receptor in HEK293 cells, a higher level of cAMP was observed in the absence of the receptor ligand, showing that Apdop1 is constitutively active. This activity was blocked by the inverse agonist flupentixol. Application of dopamine (EC50 of 35 nM) or serotonin (EC50 of 36 lM )t o Apdop1-transfected HEK293 cells further increased the level of cAMP, suggesting that the receptor is linked to the stimulatory Gs protein pathway. When expressed in cultured sensory neurons, Apdop1 immunoreactivity was observed in the cell body and neurites. Control sensory neurons responded to dopamine with a decrease in excitability mediated by a pertusis toxin-sensitive G protein. Expression of Apdop1 produced an increase in hyperpolarization in the absence of agonist and an increase in membrane excitability following stimulation by dopamine. In the presence of pertussis toxin to inhibit the Gi protein inhibitory pathway responsible for decrease in excitability mechanism, Stimulation of membrane excitability was observed. Apdop1 sensitivity to dopamine makes it a potential modulator of operant conditioning pro

Pharmacological chaperones for treating obesity

Abstract: The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and/or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

Biosensors for monitoring receptor-mediated G-protein activation

Abstract: The present invention relates to novel biosensors that are based on bioluminescence resonance energy transfer (BRET). These biosensors may be used to monitor rapid interaction and conformational changes within G protein-coupled receptor/G protein complexes and, in this way, reflect the activation status of the receptor. Advantageously, the biosensors may be used as a highly sensitive and quantitative assay for the identification of ligands (agonists, antagonists, inverse agonists, partial agonists, etc.) targeting G protein-coupled receptors (GPCRs) as well as for the analysis of the activation status of these receptors. Moreover, multiplexing different biosensors within receptors/G protein complexes allows for mapping ligand textures. Additionally, the biosensors permit the direct, real-time examination of interactions between receptors and G protein in their natural environment, the living cell.

Appréhender la complexité du vivant à l’ère des « Omiques »

Abstract: d’un être vivant, les méthodes de la génomique et de la protéomique ont provoqué un changement d’échelle. Il ne s’agit plus de formuler une hypothèse précise sur le rôle d’un gène donné et de concevoir les expériences permettant de la tester, mais plutôt d’appréhender l’implication potentielle de l’ensemble des gènes dans un ou une série de processus biologiques donnés. La valeur intrinsèque des approches non biaisées proposées par les « Omiques » découle en partie de l’humilité que nous impose la complexité du vivant. Selon les prédictions les plus classiques, les quelques 25 000 gènes du génome humain se transforment en plus de 500 000 protéines lorsqu’on prend en compte les épissages alternatifs et les modifications posttraductionnelles. Considérant que le nombre de partenaires de chaque protéine est estimé au moins à cinq, la complexité des réseaux d’interactions possibles est intimidante. Quiconque a regardé les « interactomes » engendrés par des approches protéomiques à grande échelle ne peut qu’être perplexe quant à notre capacité à formuler la meilleure hypothèse à partir de cette masse de données. Bien que ces approches aient déjà produit leurs premiers fruits en permettant, entre autres, de proposer des fonctions pour des protéines dont on ignorait jusqu’à l’existence ou de révéler des rôles inattendus pour des protéines que l’on croyait bien connaître, l’intuition et les connaissances du chercheur ne suffisent plus à exploiter de manière optimale l’ensemble des données. Une contribution de plus en plus importante d’outils informatiques est donc devenue essentielle, tant pour la gestion des données que pour la formulation d’hypothèses objectives à partir de modèles mathématiques permettant d’appréhender de façon globale des systèmes complexes. Autant par son rythme effréné que par son impact majeur sur notre conception de la recherche biomédicale, cette révolution des « omiques » est tout sauf > Le séquençage du génome de plusieurs organismes vivants incluant celui de l’homme a provoqué une véritable révolution scientifique dont nous commençons à peine à mesurer les multiples implications. Le début de l’année étant une période propice à la réflexion sur les réalisations passées, et surtout sur ce que nous réserve l’avenir, nous nous penchons dans cet Éditorial sur quelques-unes des conséquences que la révolution génomique a déjà eues et continuera d’avoir sur la recherche biomédicale. Au-delà des espoirs et des fantasmes que peut faire naître chez certains la conscience de voir exposé devant nos yeux le « code de la vie », la masse d’informations et notre capacité à appréhender « globalement » les questions biologiques ont changé de façon irrévocable notre façon de faire de la science. L’origine de cette révolution est ancrée au milieu du siècle dernier quand des pionniers tels que Chargaff, Crick, Franklin, Khoranna, Kornberg, Monod, Nirenberg, Pauling, Watson, et bien d’autres ont donné naissance à une nouvelle discipline, la biologie moléculaire. Malgré le pouvoir considérable que donnaient les nouvelles méthodes issues de cette discipline, cette première phase de la révolution était tranquille. Tranquille par son rythme, s’échelonnant sur plus d’une cinquantaine d’années, mais aussi tranquille parce qu’elle ne modifiait pas fondamentalement les étapes de la démarche scientifique qui était celle des sciences de la vie depuis près de deux siècles. Malgré la capacité de modifier, à l’échelle moléculaire, le fonctionnement des cellules jusqu’à atteindre l’intégrité ou la régulation de ses gènes, les méthodes de la biologie moléculaire ne transformaient pas notre façon de formuler une hypothèse. Celle-ci faisait toujours intervenir un savant mélange de connaissances, de raisonnement rigoureux et d’intuition qui constitue le « génie propre » du chercheur. L’approche demeurait largement réductionniste et « étapiste », proposant de mieux comprendre les rôles et fonctions d’un ou de quelques gènes à la fois. En Michel Bouvier, Gérard Friedlander