Structurally different lysophosphatidylethanolamine species stimulate neurite outgrowth in cultured cortical neurons via distinct G-protein-coupled receptors and signaling cascades.

Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that structurally different lysophosphatidylethanolamine (LPE) species, palmitoyl-LPE (16:0 LPE) and stearoyl-LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Interestingly, YM-254890, an inhibitor of Gq/11 protein, inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, pertussis toxin, an inhibitor of Gi/Go proteins, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C inhibitors on neurite outgrowth were also different. In addition, both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2, but the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.

Heterotrimeric G Proteins as Therapeutic Targets in Drug Discovery.

Heterotrimeric G proteins are molecular switches in GPCR signaling pathways and regulate a plethora of physiological and pathological processes. GPCRs are efficient drug targets, and more than 30% of the drugs in use target them. However, selectively targeting an individual GPCR may be undesirable in various multifactorial diseases in which multiple receptors are involved. In addition, abnormal activation or expression of G proteins is frequently associated with diseases. Furthermore, G proteins harboring mutations often result in malignant diseases. Thus, targeting G proteins instead of GPCRs might provide alternative approaches for combating these diseases. In this review, we discuss the biochemistry of heterotrimeric G proteins, describe the G protein-associated diseases, and summarize the currently known modulators that can regulate the activities of G proteins. The outlook for targeting G proteins to treat diverse diseases is also included in this manuscript.

Heterotrimeric G-Protein-Dependent Proteome and Phosphoproteome in Unstimulated Arabidopsis Roots.

The G-protein complex is a cytoplasmic on-off molecular switch that is set by plasma membrane receptors that activate upon binding of its cognate extracellular agonist. In animals, the default setting is the "off" resting state, while in plants, the default state is constitutively "on" but repressed by a plasma membrane receptor-like protein. De-repression appears to involve specific phosphorylation of key elements of the G-protein complex and possibly target proteins that are positioned downstream of this complex. To address this possibility, protein abundance and phosphorylation state are quantified in wild type and G-protein deficient Arabidopsis roots in the unstimulated resting state. A total of 3246 phosphorylated and 8141 non-modified protein groups are identified. It has been found that 428 phosphorylation sites decrease and 509 sites increase in abundance in the G-protein quadrupole mutant lacking an operable G-protein-complex. Kinases with known roles in G-protein signaling including MAP KINASE 6 and FERONIA are differentially phosphorylated along with many other proteins now implicated in the control of G-protein signaling. Taken together, these datasets will enable the discovery of novel proteins and biological processes dependent on G-protein signaling.

[At the heart of diabetic cardiomyopathy: Bscl2 knockout mice to investigate glucotoxicity]. / Au cœur de la cardiomyopathie diabétique - Les souris lipodystrophiques SKO comme modèle d'étude de la glucotoxicité.

Type 2 diabetes mellitus (T2DM) is a well-recognized independent risk factor for heart failure (HF). T2DM is associated with altered cardiac energy metabolism, leading to ectopic lipid accumulation and glucose overload. However, the relative contribution of these two parameters remains unclear. In order to get new insight into the mechanism involved in diabetic cardiomyopathy, the cardiac phenotype of a unique T2DM mice model has been performed: the seipin knockout mice (SKO). Cardiac phenotyping revealed a diastolic dysfunction associated with hyperglycemia in these mice with a chronic activation of the hexosamine biosynthetic pathway (HBP), suggesting a glucose overload. An inhibitor of the renal sodium/glucose cotransporter 2 (SGLT2), dapagliflozin, successfully prevented the development of cardiomyopathy in SKO mice. This is particularly relevant, given that SGLT2i treatment reduces cardiovascular event in T2DM patients. Therefore, glucose lowering appears an important therapeutic target to prevent cardiac dysfunction associated with T2DM.

Small molecules targeting heterotrimeric G proteins.

G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases.

When Escherichia coli doesn't fit the mold: A pertussis-like toxin with altered specificity.

Bacterial toxins introduce protein modifications such as ADP-ribosylation to manipulate host cell signaling and physiology. Several general mechanisms for toxin function have been established, but the extent to which previously uncharacterized toxins utilize these mechanisms is unknown. A study of an Escherichia coli pertussis-like toxin demonstrates that this protein acts on a known toxin substrate but displays distinct and dual chemoselectivity, suggesting this E. coli pertussis-like toxin may serve as a unique tool to study G-protein signaling in eukaryotic cells.

Echinacoside's nigrostriatal dopaminergic protection against 6-OHDA-Induced endoplasmic reticulum stress through reducing the accumulation of Seipin.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that echinacoside (ECH), a phenylethanoid glycoside found in Cistanche deserticola, has a protective effect against the development of PD. However, the detailed mechanisms of how ECH suppresses neuronal death have not been fully elucidated. In this study, we confirmed that ECH protects nigrostriatal neurons against 6-hydroxydopamine (6-OHDA)-induced endoplasmic reticulum stress (ERS) in vivo and in vitro. ECH rescued cell viability in damaged cells and decreased 6-OHDA-induced reactive oxygen species accumulation in vitro. It also rescued tyrosine hydroxylase and dopamine transporter expression in the striatum, and decreased α-synuclein aggregation following 6-OHDA treatment in vivo. The validated mechanism of ECH activity was the reduction in the 6-OHDA-induced accumulation of seipin (Berardinelli-Seip congenital lipodystrophy 2). Seipin has been shown to be a key molecule related to motor neuron disease and was tightly associated with ERS in a series of in vivo studies. ECH attenuated seipinopathy by promoting seipin degradation via ubiquitination. ERS was relieved by ECH through the Grp94/Bip-ATF4-CHOP signal pathway.

Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins.

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

The heterotrimeric G protein Gß1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gßγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gß1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gß1 revealed that Gß1 interacts with the PP1c α, ß, and γ1 isoforms. Purified PP1c bound to recombinant Gß1-GST protein, and PP1c co-immunoprecipitated with Gß1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gß1 complex, which correlated with an association of PP1c with phospholipase C ß3 (PLCß3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCß3. siRNA-mediated depletion of GNB1 (encoding Gß1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gßγ. Finally, disruption of PP1c-Gß1 complexes with myristoylated Gß1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gß1 protein enlists PP1c to modulate GPCR signaling in platelets.

Gß1 is required for neutrophil migration in zebrafish.

Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gß1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gßγ signaling, neutrophils failed to migrate to wound sites. Although both the Gß1 and Gß4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gß1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gß1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gß1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gß1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gß isoform in chemotaxis in vivo.

Intracellular allosteric antagonism of the CCR9 receptor.

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

Correction of the Pathogenic Alternative Splicing, Caused by the Common GNB3 c.825C>T Allele, Using a Novel, Antisense Morpholino.

The very common GNB3 c.825C>T polymorphism (rs5443) is present in approximately half of all human chromosomes. Significantly, the presence of the GNB3 825T allele has been strongly associated with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we, therefore, bioinformatically designed, using a Gene Tools(®) algorithm, a GNB3-specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ∼58% to ∼5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ∼42% to ∼95%). Our results demonstrate the potential use of a GNB3-specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

Activation of Ras-dependent signaling pathways by G(14) -coupled receptors requires the adaptor protein TPR1.

Many G(q) -coupled receptors mediate mitogenic signals by stimulating extracellular signal-regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα(q) family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα(q) family, the highly promiscuous Gα(14) can relay signals from numerous receptors. Here, we examined if Gα(14) interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα(14) QL mutant in HEK293 cells led to the formation of GTP-bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G(14) -coupled somatostatin type 2 and α(2) -adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co-immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα(14) QL than Gα(14) , suggesting that TPR1 preferentially binds to the GTP-bound form of Gα(14) . Activated Gα(14) also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA-mediated knockdown of TPR1 effectively abolished the ability of Gα(14) to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα(14) QL-induced phosphorylations of ERK and IκB kinase, it did not affect Gα(14) QL-induced stimulation of phospholipase Cß or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for Gα(14) to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways.

Identification of a GαGßγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including GαGßγ subunits, PKCα, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGßγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists GαGßγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

Replacing the rod with the cone transducin subunit decreases sensitivity and accelerates response decay.

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.

Angiotensin II-induced cyclooxygenase 2 expression in rat aorta vascular smooth muscle cells does not require heterotrimeric G protein activation.

Angiotensin II (AngII) initiates cellular effects via its G protein-coupled angiotensin 1 (AT(1)) receptor (AT(1)R). Previously, we showed that AngII-induced expression of the prostanoid-producing enzyme cyclooxygenase 2 (COX-2) was dependent upon nuclear trafficking of activated AT(1)R. In the present study, mastoparan (an activator of G proteins), suramin (an inhibitor of G proteins), 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; a specific inhibitor of phospholipase C), and sarcosine(1)-Ile(4)-Ile(8)-AngII (SII-AngII; a G protein-independent AT(1)R agonist) were used to determine the involvement of G proteins and AT(1A)R trafficking in AngII-stimulated COX-2 protein expression in human embryonic kidney-293 cells stably expressing AT(1A)/green fluorescent protein receptors and cultured vascular smooth muscle cells, respectively. Mastoparan alone stimulated release of intracellular calcium and increased COX-2 expression. Preincubation with mastoparan inhibited AngII-induced calcium signaling without altering AngII-induced AT(1A)R trafficking, p42/44 extracellular signal-regulated kinase (ERK) activation, or COX-2 expression. Suramin or U73122 had no significant effect on their own; they did not inhibit AngII-induced AT(1A)R trafficking, p42/44 ERK activation, or COX-2 expression; but they did inhibit AngII-induced calcium responses. SII-AngII stimulated AT(1A)R trafficking and increased COX-2 protein expression without activating intracellular calcium release. These data suggest that G protein activation results in increased COX-2 protein expression, but AngII-induced COX-2 expression seems to occur independently of G protein activation.

Pertussis toxin-sensitive G proteins regulate lymphoid lineage specification in multipotent hematopoietic progenitors.

Lymphoid and myeloid lineage segregation is a major developmental step during early hematopoiesis from hematopoietic stem cells. It is not clear, however, whether multipotent progenitors (MPPs) adopt a lymphoid or myeloid fate through stochastic mechanisms, or whether this process can be regulated by extracellular stimuli. In this study, we show that lymphoid lineage specification occurs in MPPs before lymphoid lineage priming, during which MPPs migrate from the proximal to the distal region relative to the endosteum of the bone marrow. Lymphoid-specified MPPs have low myeloid differentiation potential in vivo, but potently differentiate into myeloid cells in vitro. When treated with pertussis toxin, an inhibitor of G protein-coupled receptor signaling, lymphoid-specified MPPs regain in vivo myeloid potential, and their localization is dispersed in the bone marrow. These results clearly demonstrate that specific microenvironments that favorably support lymphoid or myeloid lineage development exist at structurally distinct regions in the bone marrow.

The novel inhibitor of the heterotrimeric G-protein complex, BIM-46187, elicits anti-hyperalgesic properties and synergizes with morphine.

BIM-46187 (7-[2-amino-1-oxo-3-thio-propyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo-[1,2a]-pyrazine dimer, hydrochloride) is an inhibitor of the heterotrimeric G-protein complex signalling. Since many mediators of pain act through G-protein coupled receptors, the anti-hyperalgesic effects of BIM-46187 were assessed on experimental models of pain. In addition since opioids are widely used in pain management and act through specific G-protein-coupled receptors, the effects of BIM-46187 on the analgesic properties of morphine have also been investigated. BIM-46187 elicited a dose dependent analgesic effect in the models of carrageenan-induced hyperalgesia (0.1-1 mg/kg; i.v.) and chronic constriction injury (0.3-3 mg/kg; i.v.) in rats. BIM-46187, however, up to 10 mg/kg did not modify the paw oedema induced by carrageenan excluding an anti-inflammatory effect. In addition, at these doses, the compound was not sedative as shown by the lack of effect on the motor performance in the rotarod test. The combination of BIM-46187 and morphine (ratio 1/1) resulted in an unexpected synergistic effect in the model of carrageenan-induced hyperalgesia and in the chronic constriction injury model in rats when evaluated by isobolographic analysis. This synergy allowed a reduction of at least 20 fold in the dose of each compound. Conversely, the drug combination did not increase the side effects of morphine as assessed in the rotarod test. In conclusion, BIM-46187 elicits a potent anti-hyperalgesic effect and strongly synergizes with morphine. This work highlights the role of heterotrimeric G-protein complexes in pain and supports further investigations of the use of BIM-46187 alone, or in combination with low doses of morphine, in the management of pain.