Frizzled Proteins are bona fide G Protein-Coupled Receptors

Receptors of the Frizzled family initiate Wnt ligand-dependent signaling controlling
multiple steps in organism development and highly conserved in evolution.
Misactivation of the Wnt/Frizzled signaling is cancerogenic. Frizzled receptors
launch several signaling cascades: the canonical pathway regulating beta-catenin-dependent transcription; the planar cell polarity pathway polarizing the
cytoskeleton within the epithelial plane; and the calcium pathway. Frizzled
receptors possess seven transmembrane domains and their signaling depends on
trimeric G proteins in various organisms. However, Frizzleds constitute a
distinct group within the G protein-coupled receptors (GPCR) superfamily, and
Frizzled signaling can be G protein-independent in some experimental setups, which led to concerns about the GPCR nature of Frizzled. Here we demonstrate
that human Frizzled receptors can directly bind the trimeric Go protein in a
pertussis toxin-sensitive manner. Furthermore, addition of Wnt ligands elicits
Frizzled-dependent guanine nucleotide exchange on Go. An excess of secreted
Frizzled-related protein (a Wnt antagonist) prevents Go activation, as does
pretreatment of Go with pertussis toxin. These experiments provide a biochemical
proof of the GPCR activities of Frizzled receptors and establish an in vitro assay to
monitor Frizzled activation by Wnt ligands, applicable for the high-throughput
agonist/antagonist screening

Competing Activities of Heterotrimeric G Proteins in Drosophila Wing Maturation

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development

Reggie-1/flotillin-2 promotes secretion of the long-range signalling forms of Wingless and Hedgehog in Drosophila

The lipid-modified morphogens Wnt and Hedgehog diffuse poorly in isolation yet can spread over long distances in vivo, predicting existence of two distinct forms of these mophogens. The first is poorly mobile and activates short-range target genes. The second is specifically packed for efficient spreading to induce long-range targets. Subcellular mechanisms involved in the discriminative secretion of these two forms remain elusive. Wnt and Hedgehog can associate with membrane microdomains, but the function of this association was unknown. Here we show that a major protein component of membrane microdomains, reggie-1/flotillin-2, plays important roles in secretion and spreading of Wnt and Hedgehog in Drosophila. Reggie-1 loss-of-function results in reduced spreading of the morphogens, while its overexpression stimulates secretion of Wnt and Hedgehog and expands their diffusion. The resulting changes in the morphogen gradients differently affect the short- and long-range targets. In its action reggie-1 appears specific for Wnt and Hedgehog. These data suggest that reggie-1 is an important component of the Wnt and Hedgehog secretion pathway dedicated to formation of the mobile pool of these morphogens

Drosophila GoLoco-Protein Pins is a target of G{alpha}o-mediated G Protein-coupled Receptor Signaling

G protein-coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Galpha-subunits; the resulting Galpha-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein-dependent activities, physically binding Galpha-subunits of the Galphai/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Galpha-subunits. Here we demonstrate that the poly-GoLoco-containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Galphao. We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Galphao-GTP. We further pinpoint a Lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of Galphao-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Galphao acts downstream from Frizzled and redundantly with Galphai to control the asymmetry of cell divisions

Neutrophil chemotaxis: crucial role of phosphoinositide 3-kinase γ and Rho family GTP-binding proteins

Des neutrophiles représentent le composant principal de l'immunité innée des organismes. Ils sont capables détecter des infections bactériennes, de migrer vers ces dernières afin de détruire les pathogènes envahisseurs. La vaste palette des activités des neutrophiles est contrôlée par un réseau élaboré de cascades de signalisation, qui lient des récepteurs de la surface avec l'intérieur des cellules régulant ainsi les réponses cellulaires au milieu environnant. Des agents chimiotactiques comme le fMLP ou l'interleukin-8 agissent sur les neutrophiles par l'intermédiaire de récepteurs liés aux protéines G trimériques capable d'activer la phosphoinositide 3-kinase de type γ (PI3Kγ), enzyme phosphorylant la position D3 du PIP2 et possédant une activité protéine kinase. Pour élucider le rôle de PI3Kγ dans la signalisation des neutrophiles, nous avons utilisé des souris délétées du gène codant pour la PI3Kγ (PI3Kγ -/-). Nous avons pu montré une diminution des réponses des neutrophiles PI3Kγ-/- à des agents chimiotactiques. En effet, après un "priming" avec du lipopolysaccharide, la production de radicaux oxygénés induite par le fMLP était abolie. Après induction par des agents chimiotactiques, l'adhésion et la polymérisation d'actine des neutrophiles PI3Kγ-/- étaient réduites. Enfin, le chimiotactisme des neutrophiles in vitro et in vivo était diminué, induisant ainsi une protection réduite des souris PI3Kγ -/- contre l'infection bactérienne. Ces données montrent un rôle crucial de la PI3Kγ dans les réponses inflammatoires des neutrophiles et ouvrent des perspectives quant au développement de nouvelles drogues ayant pour cible la PI3Kγ . Les réarrangements du cytosquelette d'actine sont des évènements indispensables pour des activités cellulaires comme le chimiotactisme, l'adhésion, et la phagocytose. Pour caractériser biochimiquement la signalisation conduisant à la polymérisation d'actine, nous avons mis au point un système in vitro utilisant le cytoplasme de neutrophiles humains dans lequel l'ajout de GTPγS (analogue non hydrolysable du GTP) induisait la polymérisation et la formation de réseaux d'actine. Nous avons pu montré que cet effet dépendait de l'activation des petites protéines G de la famille Rho, elle-même sous le contrôle de facteurs d'échanges nucléotidiques (GEF) membranaires, montrant ainsi leur rôle dans le contrôle du cytosquelette d'actine des neutrophiles. De façon surprenante, cette signalisation est indépendante de kinases, excluant l'implication de nombreuses cibles des protéines Rho dans l'initiation de la polymérisation d'actine. Nous avons suggéré que l'effet du GTPγS n'était pas induit par Rac et Cdc42, mais par Rho et une protéine liée à CIP4. L'ensemble de ces données ainsi que la mise au point d'un système in vitro, contribuent à une meilleure compréhension des mécanismes moléculaires impliqués dans la signalisation conduisant au cytosquelette d'actine.Neutrophils represent a key component of organism's innate immunity. They are able to sense bacterial infection, migrate to its source, and destroy the invading pathogen. The vast array of neutrophil activities is controlled by an elaborated net of signal transduction cascades, linking surface receptors with the cell interior and regulating cell responses to the extracellular milieu. Chemoattractants like fMLP or interleukin-8 activate neutrophil receptors coupled to trimeric G proteins. The latter signal to phosphoinositide 3-kinase γ (PI3Kγ), an enzyme phosphorylating D3-position of PIP2 and also possessing a protein kinase activity. To elucidate the role of PI3Kγ in neutrophil signalling, we analyzed neutrophil functions in PI3Kγ knock-out mice. Several responses to chemoattractants were impaired in PI3Kγ -/- neutrophils. After priming with lipopolysaccharide, wild type but not knock-out neutrophils responded to fMLP with a production of oxygen radicals. Chemoattractant-induced cell adhesion and actin polymerization were reduced in PI3Kγ- null neutrophils. Finally, chemotaxis of neutrophils in vitro and in vivo was impaired, resulting in reduced protection of PI3Kγ knock-out mice from infecting bacteria. These data demonstrate a crucial role of PI3Kγ in neutrophil responses in inflammation. They also put forward the enzyme as a promising target for the development of antiinflammatory drugs. Regulated in time and space rearrangements of actin cytoskeleton are crucial to exert such cell activities as chemotaxis, adhesion, and phagocytosis. To characterize biochemically signalling to actin polymerization, we designed a cell-free system from the cytosol of human neutrophils. In this system addition of GTPγS, a non-hydrolyzable analogue of GTP, induced massive actin polymerization and cross-linking. This effect was due to constitutive activation of small Rho family GTP-binding proteins, proving their role in the control of neutrophil actin cytoskeleton. Rho-protein induced actin polymerization was shown to require a plasma membrane-associated guanine nucleotide exchange factor(s). It was also shown to be exerted via a kinase-independent mechanism, excluding a multitude of proposed downstream targets of Rho proteins in the induction of actin polymerization in the neutrophil cytosol. We suggested that Rac and Cdc42 could not be the proteins mediating the effect of GTPγS. In contrast, we found that Rho and a CIP4-binding protein could initiate actin polymerization. These data are an important contribution to the molecular dissecting of cell signalling to actin cytoskeleton, and the established cell-free system will provide more insights into the mechanisms of cell activation

Improved approaches to channel capacity estimation discover compromised GPCR signaling in diverse cancer cells

Summary: Intracellular signaling orchestrates an organism’s development and functioning and underlies various pathologies, such as cancer, when aberrant. A universal cell signaling characteristic is channel capacity — the measure of how much information a given transmitting system can reliably transduce. Here, we describe improved approaches to quantify GPCR signaling channel capacity in single cells, averaged across cell population. We assess the channel capacity based on distribution of residuals by the cellular response amplitude. We further develop means to handle irregularly responding cancer cells using the integral values of their response to different agonist concentrations. These approaches enabled us to analyze, for the first time, channel capacity in single cancer cells. A universal feature emerging for different cancer cell types is a decreased channel capacity of their GPCR signaling. These findings provide experimental validation to the hypothesis that cancer is an information disease, bearing importance for basic cancer biology and anticancer drug discovery

Clofazimine: A journey of a drug

Among different strategies to develop novel therapies, drug repositioning (aka repurposing) aims at identifying new uses of an already approved or investigational drug. This approach has the advantages of availability of the extensive pre-existing knowledge of the drug’s safety, pharmacology and toxicology, manufacturing and formulation. It provides advantages to the risk-versus-rewards trade-off as compared to the costly and time-consuming de novo drug discovery process. Clofazimine, a red-colored synthetic derivative of riminophenazines initially isolated from lichens, was first synthesized in the 1950 s, and passed through several phases of repositioning in its history as a drug. Being initially developed as an anti-tuberculosis treatment, it was repurposed for the treatment of leprosy, prior to re-repositioning for the treatment of multidrug-resistant tuberculosis and other infections. Since 1990 s, reports on the anticancer properties of clofazimine, both in vitro and in vivo, started to appear. Among the diverse mechanisms of action proposed, the activity of clofazimine as a specific inhibitor of the oncogenic Wnt signaling pathway has recently emerged as the promising targeting mechanism of the drug against breast, colon, liver, and other forms of cancer. Seventy years after the initial discovery, clofazimine’s journey as a drug finding new applications continues, serving as a colorful illustration of drug repurposing in modern pharmacology