Vladimir L. Katanaev

Trimeric G Protein-Dependent Frizzled Signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. Here, we show roles for the Galpha(o) subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly transduces Fz signals from the membrane to downstream components.

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.

Creation of nanostructures to study the topographical dependency of protein adsorption

Abstract Nanostructures of sizes comparable to protein dimensions are created on Si and Ti surfaces by local anodic oxidation (LAO) using the atomic force microscope (AFM). The characterization of the surface by X-ray photoelectron spectroscopy (XPS) reveals that this method assures a modification of the topography of the surface without a change of its chemical composition. Surfaces structured by LAO therefore represent ideal systems to study the dependence of protein adsorption on topography. We are able to visualize the created nanostructures with an AFM and successively adsorb the proteins in situ, rinse and image the new surface. The densities of adsorbed proteins on the nanostructured and neat surfaces are compared and we find that the protein arrangement depends on the underlying nanostructures, showing that proteins can “sense” the topography of surfaces at the nanometer scale. This result can be considered as the nanoscale analogous of the adsorption found for cell systems on micrometer structures.

Signal transduction in neutrophil chemotaxis.

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cβ, phosphoinositide 3-kinase γ, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rhofamily GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the “compass” of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.

Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.

Targeting the Wnt pathways for therapies

The Wnt/β-catenin signaling pathway is crucial in animal development from sponges to humans. Its activity in the adulthood is less general, with exceptions having huge medical importance. Namely, improper activation of this pathway is carcinogenic in many tissues, most notably in the colon, liver and the breast. On the other hand, the Wnt/β-catenin signaling must be re-activated in cases of tissue damage, and insufficient activation results in regeneration failure and degeneration. These both medically important implications are unified by the emerging importance of this signaling pathway in the control of proliferation of various types of stem cells, crucial for tissue regeneration and, in case of cancer stem cells – cancer progression and relapse. This article aims at briefly reviewing the current state of knowledge in the field of Wnt signaling, followed by a detailed discussion of current medical developments targeting distinct branches of the Wnt pathway for anti-cancer and pro-regeneration therapies.

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. We and others have previously demonstrated that Frizzled proteins initiating the intracellular signaling are typical G protein–coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. Here we show that the α‐subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. Gαo physically binds Axin and re‐localizes it to the plasma membrane. Furthermore, Gαo suppresses Axins inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of Gαo with Axin critically depends on the RGS domain of the latter. Additionally, we show that the βγ‐component of Go can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double—direct and indirect—impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal. Developmental Dynamics 239:168–183, 2010.

Protein adsorption on topographically nanostructured titanium

We study the protein adsorption on surfaces in order to investigate their predominant role in biocompatibility. Nanostructures are created by local anodic oxidation on titanium using the atomic force microscope. A remarkable specificity of the actin filament adsorption on the nanostructure height is noticed. F-actin is observed to have a low adsorption on nanostructures of a height of 4 nm and the adsorbed proteins appear to be randomly oriented. In contrast high protein adsorption is observed for structure height between 1 and 2 nm, moreover the filaments adsorb preferentially parallel to the nanostructured pattern.

A Direct and Functional Interaction Between Go and Rab5 During G Protein–Coupled Receptor Signaling

Regulation of a small GTPase by a heterotrimeric G protein determines the outcome of signaling from the receptor Frizzled. Internal Decisions Frizzled (Fz) proteins are receptors that trigger two different types of signaling pathways. The first, canonical signaling, requires that Fz be bound by its ligand Wnt and results in the expression of target genes. The second, planar cell polarity (PCP), regulates cell position in tissues, does not seem to require Wnt, and depends on the subcellular localization of Fz. What drives the “decision” for Fz to trigger canonical rather than PCP signaling is unclear. By examining protein-protein interactions in vitro and in Drosophila wings, Purvanov et al. showed that the heterotrimeric G protein Go, which is downstream of Fz, bound to and activated Rab5, which was required for the endocytosis of Fz. Regulation of the subsequent trafficking of Fz determined the signaling outcome, with interactions of Fz with Rab4 and Rab11, which mediate recycling of Fz and other receptors, disrupting canonical signaling and promoting PCP signaling. Hence, these pathways might be mutually exclusive. Rab5 is a small guanosine triphosphatase (GTPase) that regulates the early stages of endocytosis and is conserved in eukaryotes. Rab5 regulates the internalization of receptors and other membrane-associated signaling proteins. The function of Rab5 in these processes is considered relatively passive, so that the endocytic capacity of Rab5 is used during, for example, β-arrestin–dependent internalization of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs). Direct recruitment or activation of Rab5 by the components of these signaling pathways has not been reported. Here, we demonstrate an interaction of Drosophila Rab5 and an immediate transducer of GPCR signaling, the G protein Go, in vitro and in vivo. Rab5 and Go bound to each other as purified proteins, as well as in fly extracts. In cellular assays, Go led to Rab5 activation and endosome fusion. We further showed that the Go-Rab5 interaction functioned in Drosophila planar cell polarity and Wingless signal transduction, pathways initiated by GPCRs of the Frizzled (Fz) family. Additionally, the recycling Rab GTPases Rab4 and Rab11 functioned in Fz- and Go-mediated signaling to favor planar cell polarity over canonical Wingless signaling. The interplay between heterotrimeric G proteins and Rab GTPases controlled receptor internalization, revealing a previously uncharacterized regulatory mechanism in GPCR signaling.