Heterotrimeric Gi Proteins Research Articles

Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K+ currents in cardiomyocytes and prolongs cardiac action potential duration

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias.

A single amino acid substitution in CXCL12 confers functional selectivity at the beta-arrestin level.

CXCL12/CXCR4 axis relies on both heterotrimeric Gi protein and β-arrestin coupling to trigger downstream responses. G protein activation allows for calcium flux, chemotaxis and early extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation, whereas β-arrestin recruitment leads to late signaling, receptor desensitization and internalization. Together they may regulate the balance between transactivation and transinhibition of epithelial growth factor receptor 1 (HER1). Since we have previously noted significant differences between CXCL12 and its structural variant [N33A]CXCL12 in CXCR4 signaling, we sought to better characterize them by performing cAMP inhibition and β-arrestin recruitment assays, as well as functional tests that separately investigate G protein and β-arrestin-induced responses. [N33A]CXCL12 showed reduced potency both in Gαi coupling and β-arrestin recruitment as compared to the wild type chemokine, acting as an unbiased ligand. While these findings translated into reduced potency within Gαi-dependent functions, β-arrestin-dependent modules were affected in a more peculiar way. Unlike CXCL12, the mutant analogue did not restore HB-EGF-stimulated HER1 from CXCR4-induced transinhibition, and did not trigger the late wave of ERK1/2 phosphorylation. Instead, CXCR4 internalization was not impaired upon [N33A]CXCL12 stimulation. These differences highlight the novel opportunity to dissect CXCL12 signaling within the β-arrestin layer, in which the mutant chemokine clearly favors the internalization module over the other pathways. Such functional selectivity has an impact on HER1 activation status and may play a relevant part in the crosstalk between tyrosine kinase and seven transmembrane receptors.

Growth inhibition of human breast carcinoma cells by overexpression of regulator of G-protein signaling 4.

Breast cancer remains the second largest cause of mortality in women with cancer and does not respond well to conventional therapies. Regulator of G-protein signaling 4 (RGS4) is a GTPase-activating protein of the heterotrimeric Gq and Gi proteins. Altered levels of RGS4 are reportedly linked with several human diseases, including cancer. The present study investigated whether overexpression of RGS4 inhibited the growth of human breast cancer cells. Protein expression was investigated by western blot analysis. Cell viability and apoptosis were analyzed by MTT assay and flow cytometric analysis, respectively. Cell cycle analysis was performed using propidium iodide staining in order to examine the anti-proliferative function of increased RGS4 levels. Next, changes in the expression levels of G2/M cell cycle-related proteins were examined. Overexpression of RGS4 led to the upregulation of phosphorylayed (p)-Ser216 cell division cycle (Cdc)25C and p-Tyr15 Cdc2. Importantly, MG132-induced proteasome blockade prevented degradation of RGS4. Suppression of proliferation was associated with G2/M-phase cell cycle arrest. Furthermore, enhanced endogenous RGS4 protein levels significantly inhibited breast cancer cell growth, which was reversed by a pharmacological inhibitor of RGS4. Taken together, these results suggest that overexpression of RGS4 in human breast cancer cells by molecular means may offer a potential therapeutic approach.

Inhibition of TRPM3 Ion Channels by G-Protein Beta-Gamma Subunits

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2+ permeable cation channel activated by heat, and chemical activators such as pregnenolone sulphate and CIM0216. Here we show that activation of receptors coupled to heterotrimeric Gi proteins inhibits TRPM3 currents in a mammalian expression system. This inhibition was alleviated by co-expression of proteins that act as sinks for the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, but not a constitutively active mutant of Gαo inhibited pregnenolone sulphate-induced TRPM3 currents. TRPM3 was also inhibited by purified Gβγ proteins applied to excised inside out patches, indicating a direct effect. Baclofen and somatostatin, agonists of Gi coupled receptors, inhibited Ca2+ signals induced by pregnenolone sulphate and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data show that Gi-coupled receptors regulate TRPM3 channels via direct inhibition by Gβγ.

Identification of residues crucial for the interaction between human neuroglobin and the α-subunit of heterotrimeric Gi protein.

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits the decrease in cAMP concentration, leading to protection against cell death. In the present study, we used an eukaryotic expression vector driving high-level expression of human wild-type Ngb or Ngb mutants that either exhibit or lack GDI activities in human cells. We demonstrate that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. We further demonstrate that Glu53, Glu60, and Glu118 of human Ngb are crucial for both the neuroprotective activity and interaction with Gαi1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gαi1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gαi1.

Biochemical characterization of a heterotrimeric Gi-protein activator peptide designed from the junction between the intracellular third loop and sixth transmembrane helix in the m4 muscarinic acetylcholine receptor

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors (GPCRs) that are activated by acetylcholine released from parasympathetic nerves. The mAChR family comprises 5 subtypes, m1–m5, each of which has a different coupling selectivity for heterotrimeric GTP-binding proteins (G-proteins). m4 mAChR specifically activates the Gi/o family by enhancing the guanine nucleotide exchange factor (GEF) reaction with the Gα subunit through an interaction that occurs via intracellular segments. Here, we report that the m4 mAChR mimetic peptide m4i3c(14)Gly, comprising 14 residues in the junction between the intracellular third loop (i3c) and transmembrane helix VI (TM-VI) extended with a C-terminal glycine residue, presents GEF activity toward the Gi1 α subunit (Gαi1). The m4i3c(14)Gly forms a stable complex with guanine nucleotide-free Gαi1 via three residues in the VTI(L/F) motif, which is conserved within the m2/4 mAChRs. These results suggest that this m4 mAChR mimetic peptide, which comprises the amino acid of the mAChR intracellular segments, is a useful tool for understanding the interaction between GPCRs and G-proteins.

A Conserved Phenylalanine as a Relay between the α5 Helix and the GDP Binding Region of Heterotrimeric Gi Protein α Subunit

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.

Role of Go/i subgroup of G proteins in olfactory signaling of Drosophila melanogaster

Intracellular signaling in insect olfactory receptor neurons remains unclear, with both metabotropic and ionotropic components being discussed. Here, we investigated the role of heterotrimeric Go and Gi proteins using a combined behavioral, in vivo and in vitro approach. Specifically, we show that inhibiting Go in sensory neurons by pertussis toxin leads to behavioral deficits. We heterologously expressed the olfactory receptor dOr22a in human embryonic kidney cells (HEK293T). Stimulation with an odor led to calcium influx, which was amplified via calcium release from intracellular stores. Subsequent experiments indicated that the signaling was mediated by the Gβγ subunits of the heterotrimeric Go/i proteins. Finally, using in vivo calcium imaging, we show that Go and Gi contribute to odor responses both for the fast (phasic) as for the slow (tonic) response component. We propose a transduction cascade model involving several parallel processes, in which the metabotropic component is activated by Go and Gi, and uses Gβγ.

Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis

The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.

Defective Macrophage Migration in Gαi2- but Not Gαi3-Deficient Mice

Various heterotrimeric G(i) proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gα(i) proteins in these processes, we analyzed mice genetically deficient in Gα(i2) or Gα(i3). First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gα(i2) but not Gα(i3). Gα(i2)-deficient but not wild-type or Gα(i3)-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gα(i2) had no effect on Gα(i)-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gα(i)-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gα(i2). Furthermore, knockdown of Gα(i2) caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gα(i2) but not Gα(i3). These results indicate that Gα(i2), albeit redundant to Gα(i3) in some macrophage activation processes, clearly exhibits a Gα(i) isoform-specific role in the regulation of macrophage migration.

Evolving insights regarding mechanisms for the inhibition of insulin release by norepinephrine and heterotrimeric G proteins

Norepinephrine has for many years been known to have three major effects on the pancreatic β-cell which lead to the inhibition of insulin release. These are activation of K(+) channels to hyperpolarize the cell and prevent the gating of voltage-dependent Ca(2+) channels that increase intracellular Ca(2+) concentration ([Ca(2+)](i)) and trigger release; inhibition of adenylyl cyclases, thus preventing the augmentation of stimulated insulin release by cyclic AMP; and a "distal" effect that occurs downstream of increased [Ca(2+)](i) to inhibit exocytosis. All three are mediated by the pertussis toxin (PTX)-sensitive heterotrimeric Gi and Go proteins. The distal inhibitory effect on exocytosis is now known to be due to the binding of G protein βγ subunits to the synaptosomal-associated protein of 25 kDa (SNAP-25) on the soluble NSF attachment protein receptor (SNARE) complex. Recent studies have uncovered two more actions of norepinephrine on the β-cell: 1) retardation of the refilling of the readily releasable granule pool after it has been discharged, an action that is mediated by Gαi(1) and/or Gαi(2); and 2) inhibition of endocytosis that is mediated by Gz. Of importance also are new findings that Gαo regulates the number of docked granules in the β-cell, and that Gαo(2) maintains a tonic inhibitory influence on secretion. The latter provides another explanation as to why PTX, which blocks the effect of Gαo(2), was initially called "islet activating protein." Finally, there is clear evidence that overexpression of α(2A)-adrenergic receptors in β-cells can cause type 2 diabetes.

Novel Roles for the E3 Ubiquitin Ligase Atrophin-interacting Protein 4 and Signal Transduction Adaptor Molecule 1 in G Protein-coupled Receptor Signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

CB2 Cannabinoid Receptors Promote Neural Progenitor Cell Proliferation via mTORC1 Signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

Heterotrimeric Gi Proteins Link Hedgehog Signaling to Activation of Rho Small GTPases to Promote Fibroblast Migration

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a G(i) protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-G(i) coupling in non-canonical Hh signaling.

Abstract 2390: Serotonin signal transduction in human endothelial cells

Abstract Angiogenesis is a highly regulated biological complex process, which consist of endothelial cells proliferation, migration, and tubulogenesis as key cellular steps to form new blood capillaries from preexisting vessels. Imbalance in angiogenesis contributes to a variety of human diseases. Tumor growth depends on angiogenesis and antiangiogenic agents have been developed as effective anti-cancer medicine. Recently, we have demonstrated that serotonin (5-HT), a well-recognized neurotransmitter, has a promoting effect on endothelial cells proliferation, migration and tubulogenesis, suggesting serotonin plays a role in regulation of angiogenesis. To study the role of serotonin in angiogenesis, we examined the expression of serotonin receptor isoforms in human endothelial cells. Real time PCR studies have showed that, serotonin receptor 1B (HTR1B), 1D (HTR1D), 1A (HTR1A) and 2B (HTR2B) are expressed in primary human endothelial cell lines. Blocking serotonin 1B receptor had significant inhibitory effect on endothelial cells proliferation and tubulogenesis. We have further explored the 1B-mediated signal transduction in endothelial cells. The results have revealed that the serotonin 1B receptor as a G protein-coupled receptor (GPCR) is coupled to Gi protein and decreased intracellular cAMP level upon its receptor binding. Both serotonin and 1B receptor agonist were able to activate ERK, p38, Akt, RhoA, Rac1 and PYK2/FAK signaling in endothelial cells. In contrast, a selective antagonist for serotonin 1B receptor blocks the serotonin-stimulated Akt and PYK2/FAK activation. Experiments with pertussis toxin (general Gi blocker) further demonstrated that heterotrimeric Gi-protein is required for the activation of Akt and PYK2/FAK by serotonin. These findings suggest that Akt and PYK2/FAK are involved in signaling transduction in serotonin-activated endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2390.

Purification and Identification of G Protein-coupled Receptor Protein Complexes under Native Conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.

Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles

The mechanism by which the Src family of protein-tyrosine kinases (SFKs) regulate mitogenesis and morphological changes induced by platelet-derived growth factor (PDGF) is not well known. The cholesterol-enriched membrane microdomains, caveolae, regulate PDGF receptor signalling in fibroblasts and we examined their role in SFK functions. Here we show that caveolae disruption by membrane cholesterol depletion or expression of the dominant-negative caveolin-3 DGV mutant impaired Src mitogenic signalling including kinase activation, Myc gene induction and DNA synthesis. The impact of caveolae on SFK function was underscored by the capacity of Myc to overcome mitogenic inhibition as a result of caveolae disruption. Using biochemical fractionation we show that caveolae-enriched subcellular membranes regulate the formation of PDGF-receptor-SFK complexes. An additional pool of PDGF-activated SFKs that was insensitive to membrane cholesterol depletion was characterised in non-caveolae fractions. SFK activation outside caveolae was linked to the capacity of PDGF to induce F-actin rearrangements leading to dorsal ruffle formation. Inhibition of phospholipase C gamma (PLCgamma), sphingosine kinase and heterotrimeric Gi proteins implicates a PLC gamma-sphingosine-1-phosphate-Gi pathway for PDGF-induced SFK activation outside caveolae and actin assembly. In addition, the cytoplasmic tyrosine kinase Abl was identified as an important effector of this signalling cascade. We conclude that PDGF may stimulate two spatially distinct pools of SFKs leading to two different biological outcomes: DNA synthesis and dorsal ruffle formation.

??-ARRESTINS REGULATE LIPOPOLYSACCHARIDE (LPS)-INDUCED SIGNALING AND PROINFLAMMATORY GENE EXPRESSION

Previous studies have implicated a role of heterotrimeric Gi proteins in signaling leading to inflammatory mediator production induced by LPS. β-arrestins 1 and 2 alter G protein-coupled receptors signaling. We have reported that in the β-arrestins 2 knockout mouse embryonic fibroblasts, LPS-induced IL-6 production was decreased. We hypothesized that β-arrestins 1 and/or 2 regulate LPS-induced signaling and pro-inflammatory mediator gene expression. siRNA depletion of β-arrestins 1/2 in HEK 293 cells permanently transfected with TLR4, CD14 and MD2 and β-arrestin 2 knockout (KO) mice were employed to investigate the role of β-arrestins in LPS signaling. Our data demonstrate that siRNA induced β-arrestin 2 depletion decreased LPS-induced ERK1/2 activation (37±12%), whereas, β-arrestin 1 depletion had no effect. However, depletion of both β-arrestin 1 and 2 in HEK cells reduced LPS-induced IL-8 expression (46±2% and 39±4% respectively, Figure). LPS-induced TNFα and IL-6 production was significantly decreased (37±11%, 45±10%) in splenocytes from β-arrestin 2 KO mice compared to wild type mice. These data implicate a novel role of β-arrestins in regulating LPS-induced pro-inflammatory gene expression. (Supported by NIH GM27673 and GM67202)FigureData represent means ± SE from three independent experiments. *, p<0.05 compared to basal level; #, p<0.05 compared to LPS-stimulated groups.

Gi proteins regulate lipopolysaccharide and Staphylococcus aureus induced cytokine production but not (1(r) 3)-beta-D-glucan induced cytokine suppression

Previous studies have demonstrated that bacterial lipopolysaccharide (LPS) and heat killed Staphylococcus aureus (SA) activation of inflammatory cells depended in part upon activation of heterotrimeric Gi proteins. It has also been shown that (1 --> 3) beta-D-glucan can suppress inflammatory cell activation by microbial products although the cellular mechanism of the glucan effect remains to be clearly defined. We hypothesized that Gi proteins function as a common convergent signaling pathway for both LPS and SA leading to monocyte mediator production. Additionally, we hypothesized that soluble glucan suppresses LPS and SA induced cytokine production via Gi protein coupled signaling. Human THP-1 promonocytic cells were pretreated with pertussis toxin (PTx, 100 ng/ml or 1 microgram/ml) 6 hours prior to stimulation with LPS (10 microgram/ml) and SA (10 microgram/ml) and/or soluble glucan (10 microgram/ml). Both LPS and SA significantly (p < 0.05) induced cytokine production IL-6 > TNF alpha > IL-1 beta > GM-CSF > IL-10 > IFN gamma. The induction of these cytokines was significantly (p < 0.05) suppressed by PTx. Glucan treatment alone had no effect on cytokine production but suppressed (P < 0.05) LPS and SA induced cytokines. PTx further augmented (p > 0.05) the inhibitory effect of glucan on the LPS and SA induced cytokine expression. The data support the hypothesis that Gi proteins function as a common signaling protein for both LPS and SA induction of pro-and anti-inflammatory cytokines and that soluble glucan effectively suppresses cytokine production to the microbial stimuli. In contrast, the effect of soluble glucan on inhibiting cellular activation by LPS and SA is Gi protein independent.