Regulation of G-Protein Signaling by RKTG via Sequestration of the Gβγ Subunit to the Golgi Apparatus

Upon ligand binding, G-protein-coupled receptors (GPCRs) impart the signal to heterotrimeric G proteins composed of α, β, and γ subunits, leading to dissociation of the Gα subunit from the Gβγ subunit. While the Gα subunit is imperative for downstream signaling, the Gβγ subunit, in its own right, mediates a variety of cellular responses such as GPCR desensitization via recruiting GRK to the plasma membrane and AKT stimulation. Here we report a mode of spatial regulation of the Gβγ subunit through alteration in subcellular compartmentation. RKTG (Raf kinase trapping to Golgi apparatus) is a newly characterized membrane protein specifically localized at the Golgi apparatus. The N terminus of RKTG interacts with Gβ and tethers Gβγ to the Golgi apparatus. Overexpression of RKTG impedes the interaction of Gβγ with GRK2, abrogates the ligand-induced change of subcellular distribution of GRK2, reduces isoproterenol-stimulated phosphorylation of the β2-adrenergic receptor (β2AR), and alters β2AR desensitization. In addition, RKTG inhibits Gβγ- and ligand-mediated AKT phosphorylation that is enhanced in cells with downregulation of RKTG. Silencing of RKTG also alters GRK2 internalization and compromises ligand-induced Gβ translocation to the Golgi apparatus. Taken together, our results reveal that RKTG can modulate GPCR signaling through sequestering Gβγ to the Golgi apparatus and thereby attenuating the functions of Gβγ.Heterotrimeric G proteins are composed of distinct Gα, β, and γ subunits which relay extracellular signals from heptahelical G-protein-coupled receptors (GPCRs) to downstream effectors (16, 25, 30). Gα binds Gβγ when Gα is bound with GDP but dissociates from Gβγ after GDP is replaced with GTP upon activation of GPCRs by extracellular ligand (25). Under physiologic conditions, the Gβ and Gγ subunits form a dimer in which the two subunits are not separable (10, 30). Although Gα is the primary protein that transmits the signal of GPCRs to specific intracellular effectors, such as adenylyl cyclase and phospholipase C, emerging evidence has indicated that Gβγ is able to regulate GPCR signaling through interacting with GPCRs, the Gα subunit, and downstream effectors (30). Predominantly, Gβγ is able to directly interact with and affect the functions of a variety of membrane and intracellular effectors, such as ion channels, adenylyl cyclase, G-protein-coupled receptor kinases (GRKs), and phosphatidylinositol 3-kinase (PI3K) (30). The current model of Gβγ-mediated signaling restricts it mostly to the plasma membrane (PM) (30). In the case of membrane-bound effectors, such as adenylyl cyclases or GIRK channels, Gβγ regulates the activities of these transmembrane proteins through conformational alteration. In the case of cytosolic proteins such as PLCβ2 or GRK2, whose substrates are localized to PM, Gβγ regulates their activity by recruiting the proteins to PM. The activity of Gβγ is primarily regulated by GPCR and Gα, in which GPCR activation leads to conformational changes of Gα. Such change causes replacement of Gα-bound GDP with GTP and release of Gβγ from the heterotrimeric G proteins. The activity of Gβγ could also be regulated by interacting with cytosolic proteins such as RACK1 (7). However, how Gβγ-mediated signaling is regulated in a spatial manner via subcellular compartmentation is largely unknown.GRK2 is a member of a family of GRKs that can phosphorylate the agonist-occupied GPCRs (4). Specific phosphorylation of activated receptors is associated with a decreased responsiveness of GPCR to prolonged stimulation by the agonist, also known as desensitization (15, 26). Gβγ regulates the activities of GRK2 and GRK3 toward several GPCRs (9). In cooperation with phosphatidylinositol 4,5-bisphosphate, Gβγ binds to the pleckstrin homology (PH) domain of GRK2 and recruits GRK2 to PM, in which it phosphorylates activated GPCRs (18, 30). The crystallographic structure of GRK2 in complex with Gβ1γ2 has been solved (20, 32). On the other hand, AKT is an intracellular target of PI3K and plays a critical role in cell growth, proliferation, and survival. It has been reported that Gβγ could activate AKT in a PI3K-dependent fashion (5), and Gβγ could mediate AKT activation at endosomes (13). Recent data also indicate that the p110β subunit of PI3K signals downstream of GPCR, and the AKT activation mediated by p110β is G protein dependent (14, 17).PAQR3 is a member of the progestin and adipoQ receptor (PAQR) family, and the members of this family are predicted to have seven transmembrane domains similar to GPCRs (31). Recently, we demonstrated that PAQR3 is localized at the Golgi apparatus and is involved in the spatial regulation of Raf kinase, whereby this protein was named Raf kinase trapping to Golgi apparatus (RKTG) (12). Biochemical analysis of RKTG suggested that its N terminus is localized on the cytoplasmic side of the Golgi membrane (21). Using the N terminus of RKTG to screen a Saccharomyces cerevisiae two-hybrid library, we determined that RKTG is able to interact with Gβ, and detailed analyses indicate that RKTG is a spatial regulator of Gβγ signaling.     

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

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HIF-1α Contributes to Proliferation and Invasiveness of Neuroblastoma Cells via SHH Signaling

The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on the proliferation, migration and invasion of neuroblastoma (NB) cells and the mechanisms involved. We here initially used the real-time polymerase chain reaction (real-time PCR), Western blotting and immunohistochemistry (IHC) to detect the expression of HIF-1α and components of the sonic hedgehog (SHH) signaling pathway in NB cells and human specimens. Subsequently, cell proliferation, migration and invasion were analyzed using the cell counting assay, wound healing assay and Transwell system in two types of human NB cell lines, SH-SY5Y and IMR32. In addition, the role of HIF-1α in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1α was significantly upregulated during NB progression and was associated with the expression of two components of SHH signaling, SHH and GLI1. We next indicated that the proliferation, migration and invasiveness of SH-SY5Y and IMR32 cells were significantly inhibited by HIF-1α knockdown, which was mediated by small interfering RNAs (siRNAs) targeting against its mRNA. Furthermore, the growth of NB cells in vivo was also suppressed by HIF-1α inhibition. Finally, the pro-migration and proliferative effects of HIF-1α could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1α in NB promotes proliferation, migration and invasiveness via SHH signaling.     

Cessation of Epithelial Bmp Signaling Switches the Differentiation of Crown Epithelia to the Root Lineage in a β-Catenin-Dependent Manner

The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial β-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial β-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/β-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/β-catenin pathways during cell fate decisions.     

From Squalene to Brassinolide： The Steroid Metabolic and Signaling Pathways across the Plant Kingdom

The plant steroid hormones, brassinosteroids （BRs）, and their precursors, phytosterols, play major roles in plant growth, development, and stress tolerance. Here, we review the impressive progress made during recent years in elucidating the components of the sterol and BR metabolic and signaling pathways, and in understanding their mecha- nism of action in both model plants and crops, such as Arabidopsis and rice. We also discuss emerging insights into the regulations of these pathways, their interactions with other hormonal pathways and multiple environmental signals, and the putative nature of sterols as signaling molecules.     

Distinct Molecular Evolutionary Mechanisms Underlie the Functional Diversification of the Wnt and TGFβ Signaling Pathways

The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer.     

Structural and Functional Analysis of the Regulator of G Protein Signaling 2-Gαq Complex

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Highlights? Two structures of the RGS2-Gα_q complex were determined by X-ray crystallography ? RGS2 binds Gα_q in a manner distinct from how other RGS proteins bind Gα_i/o ? In its distinct pose, RGS2 forms extensive contacts with the α-helical domain of Gα_q ? Helical domain contacts contribute to binding affinity and GAP potency of RGS2     

Has the Attraction of Predatory Coccinellids to Cornicle Droplets Constrained Aphid Alarm Signaling Behavior?

When attacked by a predator, aphids of many species secrete cornicle droplets, containing an alarm pheromone, that results in the dispersal of nearby conspecifics. As females are parthenogenetic, alarm signaling functions to enhance the survival of clone-mates. Enigmatically, however, aphids are physically able to, but usually do not emit alarm pheromone when initially detecting a predator, but rather signal only when captured by a predator. We hypothesized that cornicle droplets may be attractive to natural enemies and result in an increased risk of predation for the signaler, thereby selecting for prudent alarm signalers. We tested this hypothesis by investigating the olfactory cues that the multicolored Asian ladybird beetle, Harmonia axyridis Pallas, uses to locate pea aphids, Acyrthosiphon pisum. In choice tests, H. axyridis were attracted to odors from pea aphid colonies, whether feeding or not feeding on a host plant leaf, but were not attracted to cornicle droplets containing alarm pheromone. Further, individual pea aphids emitting cornicle droplets were not located more often or in a shorter period of time by beetles than aphids not emitting cornicle droplets. Thus, the cost of emitting early alarm signals is not prohibitively high in regards to the attraction of predators such as H. axyridis.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.     

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.     

The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell’s Response to LPA

Lysophosphatidic acid (LPA) mediates diverse cellular responses through the activation of at least six LPA receptors – LPA_1–6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA₁ contains a PDZ binding motif (SVV) identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA₁ but not that of other LPA receptors. LPA₁ colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA₁ to EEA1 early endosomes and promoted LPA₁ mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA₁ and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.     

How to identify and quantify the members of the Bacillus genus?

Members of the Bacillus genus are widely distributed throughout natural environments and have been studied for decades among others for their physiology, genetics, ecological functions, and applications. However, despite its prevalence in nature, the characterization and classification of Bacillus remain challenging due to its complex and ever-evolving taxonomic framework. This review addresses the current state of the Bacillus taxonomic landscape and summarizes the critical points in the development of Bacillus phylogeny. With a clear view of Bacillus phylogeny as a foundation, we subsequently review the methodologies applied in identifying and quantifying Bacillus, while also discussing their respective advantages and disadvantages.