Activators of G proteins inhibit GSK-3β and stabilize β-Catenin in Xenopus oocytes

Frizzled proteins, the receptors for Wnt ligands have seven hydrophobic transmembrane domains, a structural feature of G protein coupled receptors. Therefore a role for G proteins in the regulation of Wnt signaling has been proposed. Here I have used Xenopus oocytes to study the role of heterotrimeric G proteins in the regulation of GSK-3β and β-Catenin, two essential components of the canonical Wnt pathway. In these cells, general activators of G proteins such as GTPγ-S and AlF4⁻ increase β-Catenin stability and decrease GSK-3β mediated phosphorylation of the microtubule associated protein, Tau. Among several members of Gα proteins tested, expression of a constitutively active mutant of Gαq (GαqQL) led to a significant increase in accumulation of β-Catenin. The stabilization of β-Catenin mediated by Gαq was reversed by a Gαq specific inhibitor, Gp-antagonist 2A, but not by a specific blocking peptide for Gαs. Expression of GαqQL also inhibited GSK-3β-mediated tau phosphorylation in Xenopus oocytes. These results support a role for the Gq class of G proteins in the regulation of Wnt/β-Catenin signal transduction.     

The Repertoire of Heterotrimeric G Proteins and RGS Proteins in Ciona intestinalis

Background

Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown.

Methodology/Principal Findings

In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates.

Conclusions

The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes.     

Evidence for the involvement of Gi2 in activation of extracellular signal-regulated kinases in hepatocytes

Background

Activation of the extracellular signal-regulated kinases ERK1 and ERK2 in hepatocytes by prostaglandin (PG)F_2α was recently found to be inhibited by pertussis toxin (PTX) suggesting a role for G_i proteins.

Results

Targeting the Gi_2α expression by a specific ribozyme inhibited the PGF_2α -induced ERK1/2 activation in hepatocytes. On the other hand a non-cleaving form of the Gi_2α ribozyme did not significantly decrease the ERK1/2 activation. In ribozyme-treated cells the Gi_2α protein level was reduced, while the G_qα level was not affected thus confirming the specificity of the ribozyme.

Conclusion

The present data suggest an important role of G_i2 in PGF_2α -induced ERK1/2 signaling in hepatocytes.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G_s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G_s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G_s-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα_s subfamily regulates activity of Rho GTPases extends our understanding of Gα_s activity and establishes RhoGEF coupling as a universal Gα function.

The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein G_s is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gα_s will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed G_s can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (G_i, G_q, and G₁₂) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gα_s for adenylyl cyclase and cAMP; Gα_q for phospholipase C, phosphoinositides, and Ca²⁺; and Gα_i for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα₁₂ and Gα₁₃ were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G₁₂-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G₁₂ class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gα_q could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gα_s, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gα_s can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by Gα_sQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by G_s-coupled receptors, and not by G_q- or G_i-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gα_s, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the Gα_sQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for G_s; the Gα_s-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gα_s Q227L mutant. Considering the multitude of G_s-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new G_s-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gα_s (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gα_s interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gα_s bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα₁₂ and Gα₁₃, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gα_s with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G₁₂ and G_q via their α subunits and G_i via the Gβγ subunits (10). The demonstration that the G_s subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G₁₂, G_q, and G_i, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that G_s, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to G_s. IP₃, inositol 1,4,5-trisphosphate.

Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

PH

pleckstrin homology

DH

Dbl homology

GEF

guanine nucleotide exchange factor

PRG

PDZ-RhoGEF

GPCR

G protein–coupled receptor.

     

Activation of nuclear factor kappa B (NF-κB) by connective tissue growth factor (CCN2) is involved in sustaining the survival of primary rat hepatic stellate cells

Background

Secreted Frizzled related proteins (SFRPs) are extracellular regulators of Wnt signaling. These proteins contain an N-terminal cysteine rich domain (CRD) highly similar to the CRDs of the Frizzled family of seven-transmembrane proteins that act as Wnt receptors. SFRPs can bind to Wnts and prevent their interaction with the Frizzled receptor. Recently it has been reported that a splice variant of human Frizzled-4 (FZD4S) lacking the transmembrane and the cytoplasmic domains of Frizzled-4 can activate rather than inhibit Wnt-8 activity in Xenopus embryos. This indicates that secreted CRD containing proteins such as Frizzled ecto-domains and SFRPs may not always act as Wnt inhibitors. It is not known how FZD4S can activate Wnt/β-catenin signaling and what biological role this molecule plays in vivo.

Results

Here we report that the Xenopus frizzled-4 is alternatively spliced to give rise to a putative secreted protein that lacks the seven-transmembrane and the cytoplasmic domains. We performed functional experiments in Xenopus embryos to investigate how this novel splicing variant, Xfz4S, can modulate the Wnt/β-catenin pathway. We show that Xfz4S as well as the extracellular domain of Xfz8 (ECD8) can act as both activators and inhibitors of Wnt/β-catenin signaling dependent on the Wnt ligand presented. The positive regulation of Wnt/β-catenin signaling by the extracellular domains of Frizzled receptors is mediated by the members of low density lipoprotein receptor-related protein (LRP-5/6) that act as Wnt coreceptors.

Conclusion

This work provides evidence that the secreted extracellular domains of Frizzled receptors may act as both inhibitors and activators of Wnt signaling dependent on the Wnt ligand presented.     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

Frizzled receptors signal through G proteins

Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gα_i, Gα_q, and Gα_s proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gα_s. The Frizzled/Gα_s interaction was further established in vivo as Drosophila expressing a loss-of-function Gα_s allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gα_s heterotrimeric G proteins.     

Breast cancer cell invasion mediated by Gα12 signaling involves expression of interleukins-6 and −8, and matrix metalloproteinase-2

Background

Recent studies on the involvement of the G12 family of heterotrimeric G proteins (Gα12 and Gα13, the products of the GNA12 and GNA13 genes, respectively) in oncogenic pathways have uncovered a link between G12 signaling and cancer progression. However, despite a well characterized role of Rho GTPases, the potential role of secreted factors in the capacity of G12 signaling to promote invasion of cancer cells is just beginning to be addressed.

Methods

MDA-MB-231 and MCF10A breast cancer cell lines were employed as a model system to explore the involvement of secreted factors in G12-stimulated cell invasion. Factors secreted by cells expressing dominant-active Gα12 were identified by protein array, and their involvement in breast cancer cell invasion was assessed through both RNAi-mediated knockdown and antibody neutralization approaches. Bioinformatics analysis of the promoter elements of the identified factors suggested NF-κB elements played a role in their enhanced expression, which was tested by chromatin immunoprecipitation.

Results

We found that signaling through the Gα12 in MDA-MB-231 and MCF10A breast cancer cell lines enhances expression of interleukins (IL)-6 and ?8, and matrix metalloproteinase (MMP)-2, and that these secreted factors play a role in G12-stimulated cell invasion. Furthermore, the enhanced expression of these secreted factors was found to be facilitated by the activation of their corresponding promoters, where NF-κB seems to be one of the major regulators. Inhibition of IL-6 and IL-8, or MMP-2 activity significantly decreased Gα12-mediated cell invasion.

Conclusions

These studies confirm and extend findings that secreted factors contribute to the oncogenic potential of G12 signaling, and suggest potential therapeutic targets to control this process.

     

Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast

Background

Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose.

Results

In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the k_on (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus.

Conclusions

Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.     

Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells

Background

Nucleotide-actived P2Y receptors play critical roles in the growth of tumor cells by regulating cellular proliferation, differentiation and survival.

Results

Here we demonstrate that an avian P2Y purinoceptor (tP2YR) with unique pharmacological and signal transduction properties induces morphologic and growth transformation of rodent fibroblasts. tP2YR induced a transformed phenotype similar to the mas oncogene, a G protein-coupled receptor which causes transformation by activation of Rac-dependent pathways. tP2YR-transformed cells exhibited increased steady-state activation of Rac1 and RhoA. Like activated Rho GTPases, tP2YR cooperated with activated Raf and caused synergistic transformation of NIH3T3 cells. Our data indicate that the ability of tP2YR to cause transformation is due to its unique ability among purinergic receptors to simultaneously activate Gα_q and Gα_i. Co-expression of constitutively activated mutants of these two Gα subunits caused the same transformed phenotype as tP2YR and Mas. Furthermore, transformation by both tP2YR and Mas was blocked by pharmacological inhibition of Gα_I by pertussis toxin (PTX) indicating an essential role for Gα_i in transformation by these G-protein coupled receptors.

Conclusions

Our data suggest that coordinated activation of Gα_q and Gα_i may link the tP2YR and possibility the Mas oncogene with signaling pathways resulting in activation of Rho family proteins to promote cellular transformation.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

Background

The classic paradigm of heterotrimeric G-protein signaling describes a heptahelical, membrane-spanning G-protein coupled receptor that physically interacts with an intracellular Gα subunit of the G-protein heterotrimer to transduce signals. G-protein coupled receptors comprise the largest protein superfamily in metazoa and are physiologically important as they sense highly diverse stimuli and play key roles in human disease. The heterotrimeric G-protein signaling mechanism is conserved across metazoa, and also readily identifiable in plants, but the low sequence conservation of G-protein coupled receptors hampers the identification of novel ones. Using diverse computational methods, we performed whole-proteome analyses of the three dominant model plant species, the herbaceous dicot Arabidopsis thaliana (mouse-eared cress), the monocot Oryza sativa (rice), and the woody dicot Populus trichocarpa (poplar), to identify plant protein sequences most likely to be GPCRs.

Results

Our stringent bioinformatic pipeline allowed the high confidence identification of candidate G-protein coupled receptors within the Arabidopsis, Oryza, and Populus proteomes. We extended these computational results through actual wet-bench experiments where we tested over half of our highest ranking Arabidopsis candidate G-protein coupled receptors for the ability to physically couple with GPA1, the sole Gα in Arabidopsis. We found that seven out of eight tested candidate G-protein coupled receptors do in fact interact with GPA1. We show through G-protein coupled receptor classification and molecular evolutionary analyses that both individual G-protein coupled receptor candidates and candidate G-protein coupled receptor families are conserved across plant species and that, in some cases, this conservation extends to metazoans.

Conclusion

Our computational and wet-bench results provide the first step toward understanding the diversity, conservation, and functional roles of plant candidate G-protein coupled receptors.     

Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

Background

Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta ₂-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta₂-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.

Results

The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta₂-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta₂-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β₂-adrenergic receptor for those of Ste2 creates a Ste2/beta₂-adrenergic receptor chimera displaying insulin-stimulated internalization.

Conclusion

Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.     

A role for G proteins in plant hormone signalling?

The G protein signalling pathway is one of the most highly conserved mechanisms that enables cells to sense and respond to changes in their environment. Essential components of this are cell surface G protein-coupled receptors (GPCRs) that perceive extracellular ligands, and heterotrimeric G proteins (G proteins) that transduce information from activated GPCRs to down-stream effectors such as enzymes or ion channels. It is now clear from a range of biochemical and molecular studies that some potential G protein signalling components exist in plants. The best examples of these are the seven transmembrane receptor homologue GCR1 and the G_α (GPA1) and G_β (Gβ1) subunit homologues of heterotrimeric G proteins. G protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate G proteins in a range of signalling pathways that include the plant hormones gibberellin and auxin. Furthermore, antisense suppression of GCR1 expression in Arabidopsis leads to a phenotype that supports a role for this receptor in cytokinin signalling. This review considers the current evidence for and against functional G protein signalling pathways in higher plants and questions whether or not these might be involved in the action of certain plant hormones.     

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G_i/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-³⁵S]GTP assay and activity state-specific antibodies to GTP-bound G_i proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G_i2/3 proteins in N13 cells possibly mediated by FZD₅, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G_i2/3 proteins in mammalian cells with physiological protein stochiometry.     

Mutations on the Switch III region and the alpha3 helix of Galpha16 differentially affect receptor coupling and regulation of downstream effectors

Background

Gα₁₆ can activate phospholipase Cβ (PLCβ) directly like Gα_q. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα₁₆. Previous studies on Gα_q have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα₁₆ might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.

Results

Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα₁₆. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα₁₆ was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G₁₆ and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.

Conclusion

The integrity of the switch III region and α3 helix of Gα₁₆ is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα₁₆ to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G₁₆. The studied region was shown to bear multiple functionally important roles of G₁₆.     

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

Background

Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins G??_q, G??₁₃ and G??_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.

Results

Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by G??_i-triggered signalling as well as by G?|?-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NF?B-pathway and thereby the production of TNF-??, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by G??_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by G?|?-mediated activation of PI3kinase and JNK activation.

Conclusions

On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host??s immune response.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.