Regulation of frizzled receptor activity at the plasma membrane : an interplay of the receptor, the trimeric G protein Go, and RGS protein and a scaffolding protein Kermit

G-protein-signaling pathways convey extracellular signals inside the cells and regulate distinct physiological responses. This type of signaling pathways consists of three major components: G-protein-coupled receptors (GPCRs), heterotrimeric G proteins (G-proteins) and downstream effectors. Upon ligand binding, GPCRs activate heterotrimeric G proteins to initiate the signaling cascade. Dysfunction of GPCR signaling correlates with numerous diseases such as diabetes, nervous and immune system deficiency, and cancer. As the signaling switcher, G-proteins (Gs, Gq/11, G12/13, and Gi/o) have been an appealing topic of research for decades. A heterotrimeric G-protein is composed of three subunits, the guanine nucleotide associated a-subunit, ß and y subunits. In general, the duration of signaling is determined by the lifetime of activated (GTP bound) Ga subunits. Identification of novel communication partners of Ga subunits appears to be an attractive way to understand the machinery of GPCR signaling. In our lab, we mainly focus on Gao, which is abundantly expressed in the nervous system. Here we present two novel interacting partners of Drosophila Gao: Dhit and Kermit, identified through yeast two-hybrid screening and genetic screening respectively. Dhit is characterized by a small size with a conserved RGS domain and an N-terminal cysteine rich motif. The RGS domain possesses the GAP (GTPase activating protein) activity towards G proteins. However, we found that Dhit exerts not only the GAP activity but also the GDI (guanine nucleotide dissociation inhibitor) activity towards Gao. The unexpected GDI activity is preserved in GAIP/RGS19 - a mammalian homologue of Dhit. Further experiments confirmed the GDI activity of Dhit and GAIP/RGS19 in Drosophila and mammalian cell models. Therefore, we propose that Dhit and its mammalian homologues modulate GPCR signaling by a double suppression of Ga subunits - suppression of their nucleotide exchange with GTP and acceleration of their hydrolysis of GTP. Kermit/GEPC was first identified as a binding partner of GAIP/RGS19 in a yeast two- hybrid screen. Instead of interacting with the Drosophila homologue of GAIP/RGS19 (Dhit), Kermit binds to Gao in vivo and in vitro. The functional consequence of Kermit/Gao interaction is the regulation of localization of Vang (one of the planar cell polarity core components) at the apical membrane. Overall, my work elaborated the action of Gao with its two interaction partners in Gao- mediated signaling pathway. Conceivably, the understanding of GPCR signaling including Gao and its regulators or effectors will ultimately shed light on future pharmaceutical research. - Les voies de signalisation médiées par les protéines G transmettent des signaux extracellulaires à l'intérieur des cellules pour réguler des réponses physiologiques distinctes. Cette voie de signalisation consiste en trois composants majeurs : les récepteurs couplés aux protéines G (GPCRs), les protéines G hétérotrimériques (G-proteins) et les effecteurs en aval. Suite à la liaison du ligand, les GPCRs activent les protéines G hétérotrimériques qui initient la cascade de signalisation. Des dysfonctions dans la signalisation médiée par les GPCRs sont corrélées avec de nombreuses maladies comme le diabète, des déficiences immunes et nerveuses, ainsi que le cancer. Puisque la voie de signalisation s'active et se désactive, les protéines G (Gs, Gq/11, G12/13 et Gi/o) ont été un sujet de recherche attrayant pendant des décennies. Une protéine G hétérotrimérique est composée de trois sous-unités, la sous-unité a associée au nucléotide guanine, ainsi que les sous-unités ß et y. En général, la durée du signal est déterminée par le temps de demi-vie des sous-unités Ga activées (Ga liées au GTP). Identifier de nouveaux partenaires de communication des sous-unités Ga se révèle être un moyen attractif de comprendre la machinerie de la signalisation par les GPCRs. Dans notre laboratoire nous nous sommes concentrés principalement sur Gao qui est exprimée de manière abondante dans le système nerveux. Nous présentons ici deux nouveaux partenaires qui interagissent avec Gao chez la drosophile: Dhit et Kermit, qui ont été identifiés respectivement par la méthode du yeast two-hybrid et par criblage génétique. Dhit est caractérisé par une petite taille, avec un domaine RGS conservé et un motif N- terminal riche en cystéines. Le domaine RGS contient une activité GAP (GTPase activating protein) pour les protéines G. Toutefois, nous avons découvert que Dhit exerce non seulement une activité GAP mais aussi une activité GDI (guanine nucleotide dissociation inhibitor) à l'égard de Gao. Cette activité GDI inattendue est préservée dans RGS19 - un homologue de Dhit chez les mammifères. Des expériences supplémentaires ont confirmé l'activité GDI de Dhit et de RGS19 chez Drosophila melanogaster et les modèles cellulaires mammifères. Par conséquent, nous proposons que Dhit et ses homologues mammifères modulent la signalisation GPCR par une double suppression des sous-unités Ga - suppression de leur nucléotide d'échange avec le GTP et une accélération dans leur hydrolyse du GTP. Kermit/GIPC a été premièrement identifié comme un partenaire de liaison de RGS19 dans le criblage par yeast two-hybrid. Au lieu d'interagir avec l'homologue chez la drosophile de RGS19 (Dhit), Kermit se lie à Gao in vivo et in vitro. La conséquence fonctionnelle de l'interaction Kermit/Gao est la régulation de la localisation de Vang, un des composants essentiel de la polarité planaire cellulaire, à la membrane apicale. Globalement, mon travail a démontré l'action de Gao avec ses deux partenaires d'interaction dans la voie de signalisation médiée par Gao. La compréhension de la signalisation par les GPCRs incluant Gao et ses régulateurs ou effecteurs aboutira à mettre en lumière de futurs axes dans la recherche pharmacologique.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Pathophysiological mechanisms of catecholamine and cocaine-mediated cardiotoxicity.

Overactivation of the sympatho-adrenergic system is an essential mechanism providing short-term adaptation to the stressful conditions of critical illnesses. In the same way, the administration of exogenous catecholamines is mandatory to support the failing circulation in acutely ill patients. In contrast to these short-term benefits, prolonged adrenergic stress is detrimental to the cardiovascular system by initiating a series of adverse effects triggering significant cardiotoxicity, whose pathophysiological mechanisms are complex and only partially elucidated. In addition to the development of myocardial oxygen supply/demand imbalance induced by the sustained activation of adrenergic receptors, catecholamines can damage cardiomyocytes by fostering mitochondrial dysfunction, via two main mechanisms. The first one is calcium overload, consecutive to β-adrenergic receptor-mediated activation of protein kinase A and subsequent phosphorylation of multiple Ca(2+)-cycling proteins. The second one is oxidative stress, primarily related to the transformation of catecholamines into "aminochromes," which undergo redox cycling in mitochondria to generate copious amounts of oxygen-derived free radicals. In turn, calcium overload and oxidative stress promote mitochondrial permeability transition and cardiomyocyte cell death, both via the apoptotic and necrotic pathways. Comparable mechanisms of myocardial toxicity, including marked oxidative stress and mitochondrial dysfunction, have been reported with the use of cocaine, a common recreational drug with potent sympathomimetic activity. The aim of the current review is to present in detail the pathophysiological processes underlying the development of catecholamine and cocaine-induced cardiomyopathy, as such conditions may be frequently encountered in the clinical practice of cardiologists and ICU specialists.

Olfactory mediated interactions between Citrus aurantium Toxoptera citricida and Lysiphlebus testaceipes

The objective of this study was to establish whether there are olfactory interactions in the Lysiphlebus testaceipes Toxoptera citricida and Citrus aurantium tritrophic system. The response of male and female L. testaceipes to different odour sources of the host plant C. aurantium, the aphid host T. citricida and aphid-plant complex were investigated using a Y-tube olfactometer. Laboratory experiments were conducted by exposing individually aged male and female L. testaceipes to eight different odour treatments. Response of the parasitoids was taken after 15 min exposure to the volatiles from the different odour sources and based on their orientation to the particular chamber. Seventy percent of both male and female L. testaceipes showed high attractivity to aphid infested leaves. There was no significant difference based on age and sex of the parasitoid on their choice of odour. The organic compounds released by these combinations acted as semiochemicals in the tritrophic interactions and it is suggested that insect feeding induced attraction of the parasitoid L. testaceipes.

Anne Gangloff, Dion Chrysostome et les mythes. Hellénisme, communication et philosophie politique, París (J. Millon) 2006.

Thi book, as its author makes clear, is based on a thesis that set out initially to analyse what the myths in the works of Dio Chrysostom actually represented but as Gangloff proceeded whit her research her analysis became rather an examination of how the sophist took over and reinvented myths, adpting them to his own purposes and his own times.

Premsa gratuïta, un model de comunicació local = Free press, a local communication model

The relationship between non-institutional free press and local communication is quite particular since this type of press forms a very characteristic model of local communication, showing that advertising suffices to finance an information product addressed to a fairly well-defined readership as long as this product has a good advertising sales department and an effective distribution in its operating area. This paper discusses the present situation of the free press in Catalonia, where this phenomenon has been quite prominent. It points out the main features of this type of press and makes a review of its history, which runs from the euphoria of its early years and its expansion and consolidation, to the current crisis

Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Sporozoite-mediated hepatocyte wounding limits Plasmodium parasite development via MyD88-mediated NF-kappa B activation and inducible NO synthase expression

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-kappaB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-kappaB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-kappaB inhibitor BAY11-7082. Furthermore, no NF-kappaB activation was observed when Spect(-/-) parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-kappaB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88(-/-) mice showed no NF-kappaB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.

Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the alpha1B-adrenergic receptor.

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the alpha1B-adrenergic receptor (alpha1BAR). In this study, phosphoamino acid analysis of the phosphorylated alpha1BAR revealed that both epinephrine- and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist- as well as phorbol ester-induced phosphorylation and desensitization of the alpha1BAR. To identify the serines invoved in agonist- versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Our findings indicate that Ser394 and Ser400 were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser404, Ser408, and Ser410 were phosphorylated upon stimulation of the alpha1BAR with epinephrine. The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser404, Ser408, and Ser410, strongly suggests that these serines are the phosphorylation sites of the alpha1BAR for kinases of the GRK family. Phorbol ester-induced phosphorylation of the Ser394 and Ser400 as well as GRK2-mediated phosphorylation of the Ser404, Ser408, and Ser410, resulted in the desensitization of alpha1BAR-mediated inositol phosphate response. This study provides generalities about the biochemical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholipase C.

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its {beta}1 Subunit.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.