Céline Galés

CODA-RET reveals functional selectivity as a result of GPCR heteromerization.

Here we present a novel method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 receptor (D2R) agonist R(–)-Propylnorapomorphine (NPA) to change the Gαi conformation via the D2R protomer in the D1-D2 heteromer was enhanced 10-fold relative to that observed in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity, in which a drug acts differently at a GPCR protomer depending on the identity of the second protomer that participates in forming the signaling unit, opening the door to enhanced pharmacological specificity through targeting differences between homomeric and heteromeric signaling.

Functional Selective Oxytocin-derived Agonists Discriminate between Individual G Protein Family Subtypes

Background: The oxytocin receptor couples to multiple G proteins, leading to different physiological responses. Results: We screened for functional selective oxytocin receptor agonists and identified two analogs that activate individual Gi subunits. Conclusion: Functional selective analogs discriminate among different receptor conformations coupled to Gi proteins. Significance: These compounds will contribute to the development of selective drugs with new selectivity and therapeutic profiles. We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of Gq and all the Gi/o subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated GoA or GoB, and, with only one exception, all of the peptides that activated Gq also activated Gi2 and Gi3 but not Gi1, GoA, or GoB, indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only Gi1 or Gi3, failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual Gi/o family members. Both analogs inhibited cell proliferation, showing that a single Gi subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of Gi/o members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

Specific interactions between Epac1, β-arrestin2 and PDE4D5 regulate β-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling

β1 and β2 adrenergic receptors (βARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both βAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of βARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein β-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that β-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of β-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon β1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with β-arrestin2 following β2AR activation. Dissociation of the PDE4D5-β-arrestin2 complex allowed the recruitment of Epac1 to β2AR and induced a switch from β2AR non-hypertrophic signaling to a β1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which βAR subtypes exert opposing effects.

Specific roles of Gi protein family members revealed by dissecting SST5 coupling in human pituitary cells

Summary Despite intensive investigation over the past 20 years, the specific role played by individual Gi protein family members in mediating complex cellular effects is still largely unclear. Therefore, we investigated the role of specific Gi proteins in mediating somatostatin (SS) effects in somatotroph cells. Because our previous data showed that SS receptor type 5 (SST5) carrying a spontaneous R240W mutation in the third intracellular loop had a similar ability to inhibit intracellular cAMP levels to the wild-type protein but failed to mediate inhibition of growth hormone (GH) release and cell proliferation, we used this model to check specific receptor–G-protein coupling by a bioluminescent resonance energy transfer analysis. In HEK293 cells, wild-type SST5 stimulated the activation of G&agr;i1–3 and G&agr;oA, B, whereas R240W SST5 maintained the ability to activate G&agr;i1–3 and G&agr;oB, but failed to activate the splicing variant G&agr;oA. To investigate the role of the selective deficit in G&agr;oA coupling, we co-transfected human adenomatous somatotrophs with SST5 and a pertussis toxin (PTX)-resistant G&agr;oA (G&agr;oA(PTX-r)) protein. In PTX-treated cells, G&agr;oA(PTX-r) rescued the ability of the selective SST5 analog BIM23206 to inhibit extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation, GH secretion and intracellular cAMP levels. Moreover, we demonstrated that silencing of G&agr;oA completely abolished SST5-mediated inhibitory effects on GH secretion and ERK1/2 phosphorylation, but not on cAMP levels. In conclusion, by analysing the coupling specificity of human SST5 to individual G&agr;i and G&agr;o subunits, we identified a crucial role for G&agr;oA signalling in human pituitary cells.

Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between E/D3.49 and R3.50 within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E3.49/6.30 in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow “resistant” to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

Nanoscale structural mapping as a measure of maturation in the murine frontal cortex

Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.

Biophysical properties of cardiomyocyte surface explored by multiparametric AFM

PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolemmal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation

Significance G protein-coupled receptors (GPCRs) are one of the largest cell surface receptor family that transmit their signal through coupling to intracellular partners, such as G proteins. Receptor oligomerization has been shown to be pivotal in this signaling process. To address how oligomerization can impact on signaling in a major physiological process, dopamine signaling, we used a purified GPCR heteromer composed of the ghrelin and dopamine receptors to which we applied a variety of state-of-the-art biochemical and biophysical approaches. By doing so, we provide a direct experimental evidence for a mechanism where receptor heteromerization affects the conformation of the associated G protein. This sheds light on the way a GPCR oligomer can affect G protein activation to modulate signaling. The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

Les cardiomyocytes adultes se réveillent - Un grand pas vers la régénération cardiaque

NOUVELLES 237 Les cardiomyocytes adultes se réveillent : un grand pas vers la régénération cardiaque Céline Mias, Céline Galés 239 Contribution au développement de nouvelles thérapies contre le cancer colorectal : l’initiative CReMEC Alain Bruno 242 Hyperprolactinémie et infertilité : approche physiopathologique Charlotte Sonigo, Jacques Young, Nadine Binart 245 Rôle de l’hormone thyroïdienne T3 dans la régénération axonale chez les vertébrés supérieurs Hasan X. Avci, Frédéric Flamant, Isabelle Dusart 247 Arfaptine-1 et biogenèse des granules de sécrétion Joëlle Morvan, Helmuth Gehart, Roméo Ricci 249 La E-sélectine, un régulateur clé de la division des cellules souches hématopoïétiques et de leur résistance à la chimiothérapie Jean-Pierre Levesque, Ingrid G. Winkler 252 Contrôle du métabolisme énergétique par les peptides natriurétiques Cédric Moro 254 Rôle crucial de FGF20 et FGF9 pour le maintien des progéniteurs rénaux lors du développement rénal Cécile Jeanpierre 257 Rôle inattendu de Scl dans l’endothélium embryonnaire Amélie Montel-Hagen, Ben Van Handel, Hanna Mikkola 260 De l’importance de la localisation des récepteurs du glutamate NMDA Stéphane H.R. Oliet, Thomas Papouin 262 Production d’un pancréas fonctionnel in vivo par complémentation de blastocyste : preuve de concept chez le porc Laure Coulombel

Structural evidence for a new elaborate 3D-organization of the cardiomyocyte lateral membrane in adult mammalian cardiac tissues

AIMS This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.