Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes

Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (K(D) = 6.4 +/- 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA(1-3) receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.     

Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G(s)

There is great therapeutic interest in manipulating (either enhancing or suppressing) G protein-coupled receptor (GPCR) signal transduction. However, most current strategies are limited to pharmacological activation or blockade of receptors. Human gene therapy, including both overexpression and antisense approaches, may allow manipulation of GPCR signaling at steps distal to receptors. To fully understand the impact of such therapy, the transduction of signals between the multiple components of GPCR signaling and their interaction with other cellular molecules must be understood in the context of both normal physiology and disease. Defining the stoichiometric relationship among multiple components of GPCR signaling is a first step. We summarize data showing the substantial excess of G(alphas) relative to both beta-adrenergic receptors and adenylyl cyclase. A predominant idea regarding signaling via GPCRs has for over 20 years emphasized the concept of random movement and collision ("collision coupling") of proteins within the lipid bilayer of the plasma membrane. This notion does not readily account for the rapidity and fidelity of signal transduction by the multiple components involved in GPCR-G protein-effector systems, especially considering the low abundance of these proteins in cells. Recently, many components involved in signal transduction by GPCRs have been shown to exist primarily in microdomains of the plasma membrane, in particular, caveolae. These and other structures may serve to compartmentalize signals, thereby optimizing signal transduction between an agonist and specific effectors. The formation, organization, and maintenance of such structures may prove to be altered in disease states associated with disregulated signaling. In addition, we speculate that identification of genetic polymorphisms of and therapy targeted to components that are critical for determining efficacy (e.g., effectors such as adenylyl cyclase) will provide important future therapeutic strategies.     

The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain

It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.     

G protein-coupled receptors of the hypothalamic-pituitary-gonadal axis: a case for Gnrh, LH, FSH, and GPR54 receptor ligands

The hypothalamic-pituitary-gonadal (HPG) axis, important in reproduction and sex hormone-dependent diseases, is regulated by a number of G protein-coupled receptors. The recently "deorphanized" GPR54 receptor activated by the peptide metastin is thought to be the key regulator of the axis, mainly by releasing gonadotropin-releasing hormone (GnRH) from the hypothalamus. The latter decapeptide, through the activation of the GnRH receptor in the anterior pituitary, causes the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which subsequently activate their respective receptors on the gonadotrope cells. In this review we will discuss the small molecule agonists and antagonists that are currently being developed to intervene with the action of these four receptors. For GnRH receptors, 14 different chemical classes of non-peptidic antagonists have been reported, while for the LH receptor three classes of agonists have been described. Both agonists and antagonists have been introduced for the FSH receptor. Recently, the first non-peptidic agonist for GPR54 was reported.     

Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.     

Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts

G protein-coupled receptors (GPCRs) play a key role in regulating bone remodeling. Whether GPCRs exert anabolic or catabolic osseous effects may be determined by the rate of receptor desensitization in osteoblasts. Receptor desensitization is largely mediated by direct phosphorylation of GPCR proteins by a family of enzymes termed GPCR kinases (GRKs). We have selectively manipulated GRK activity in osteoblasts in vitro and in vivo by overexpressing a GRK inhibitor. We found that expression of a GRK inhibitor enhanced parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor-stimulated cAMP generation and inhibited agonist-induced phosphorylation of this receptor in cell culture systems, consistent with attenuation of receptor desensitization. To determine the effect of GRK inhibition on bone formation in vivo, we targeted the expression of a GRK inhibitor to mature osteoblasts using the mouse osteocalcin gene 2 (OG2) promoter. Transgenic mice demonstrated enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker dexoypyridinoline. Both osteoprotegrin and OPG ligand mRNA levels were altered in calvaria of transgenic mice in a pattern that would promote osteoclast activation. The predominant effect of the transgene, however, was anabolic, as evidenced by an increase in bone density and trabecular bone volume in the transgenic mice compared with nontransgenic littermate controls.     

Insulin receptor substrate 2 plays a crucial role in beta cells and the hypothalamus

We previously demonstrated that insulin receptor substrate 2 (Irs2) KO mice develop diabetes associated with hepatic insulin resistance, lack of compensatory beta cell hyperplasia, and leptin resistance. To more precisely determine the roles of Irs2 in beta cells and the hypothalamus, we generated beta cell-specific Irs2 KO and hypothalamus-specific Irs2 knockdown (betaHT-IRS2) mice. Expression of Irs2 mRNA was reduced by approximately 90% in pancreatic islets and was markedly reduced in the arcuate nucleus of the hypothalamus. By contrast, Irs2 expression in liver, muscle, and adipose tissue of betaHT-IRS2 mice was indistinguishable from that of control mice. The betaHT-IRS2 mice displayed obesity and leptin resistance. At 4 weeks of age, the betaHT-IRS2 mice showed normal insulin sensitivity, but at 8 and 12 weeks, they were insulin resistant with progressive obesity. Despite their normal insulin sensitivity at 8 weeks with caloric restriction, the betaHT-IRS2 mice exhibited glucose intolerance and impaired glucose-induced insulin secretion. beta Cell mass and beta cell proliferation in the betaHT-IRS2 mice were reduced significantly at 8 and 12 weeks but not at 10 days. Insulin secretion, normalized by cell number per islet, was significantly increased at high glucose concentrations in the betaHT-IRS2 mice. We conclude that, in beta cells and the hypothalamus, Irs2 is crucially involved in the regulation of beta cell mass and leptin sensitivity.     

染料木黄酮不是通过雌激素受体促进成骨细胞增殖

目的：探讨染料木黄酮对成骨细胞活性的影响及其可能机制。为染料木黄酮防治骨质疏松提供理论依据。 方法：实验于2001—05／2003-01在卫生学环境医学研究所完成。无菌条件下用胰蛋白酶和Ⅱ型胶原酶分步消化乳鼠颅盖骨获得成骨细胞。传代后细胞用于实验。染料木黄酮组分别加入10^5 ～10^-7 mol／L染料木黄酮，对照组以吐温20为对照。分别用噻唑蓝比色法和^3H-胸腺嘧啶掺入法测定成骨细胞增殖和DNA合成。用反转录-聚合酶链式反应和Western blot测定雌激素受体mRNA和蛋白表达。 结果：①成骨细胞培养基中加入（10^-5～10^-6mol／L）染料木黄酮，培养48h和72h后噻唑蓝的吸光度值与对照组相比均明显升高[培养48h，（039&;#177;0．03），（0．45&;#177;0．03）,（0．46&;#177;0．02），（0．19&;#177;0．05）；培养72h：（0．29&;#177;0．04），（032&;#177;0．02），（0．37&;#177;0．02），（0.15&;#177;0．04）]。②染料木黄酮组^3H-胸腺嘧啶掺入量均显著增加[染料木黄酮组为（101．20&;#177;10．06），（844．60&;#177;366．90），（512．20&;#177;197．6）；对照组为（68．67&;#177;10．39）]未发现成骨细胞雌激素受体基因和蛋白表达。 结论：染料木黄酮不是通过促进雌激素受体基因和蛋白的表达来促进成骨细胞增殖。     

染料木黄酮不是通过雌激素受体促进成骨细胞增殖

目的:探讨染料木黄酮对成骨细胞活性的影响及其可能机制,为染料木黄酮防治骨质疏松提供理论依据。方法:实验于2001-05/2003-01在卫生学环境医学研究所完成。无菌条件下用胰蛋白酶和Ⅱ型胶原酶分步消化乳鼠颅盖骨获得成骨细胞,传代后细胞用于实验。染料木黄酮组分别加入10-5～10-7mol/L染料木黄酮,对照组以吐温20为对照。分别用噻唑蓝比色法和3H-胸腺嘧啶掺入法测定成骨细胞增殖和DNA合成,用反转录-聚合酶链式反应和Westernblot测定雌激素受体mRNA和蛋白表达。结果:①成骨细胞培养基中加入(10-5～10-6mol/L)染料木黄酮,培养48h和72h后噻唑蓝的吸光度值与对照组相比均明显升高[培养48h:(0.39±0.03),(0.45±0.03),(0.46±0.02),(0.19±0.05);培养72h:(0.29±0.04),(0.32±0.02),(0.37±0.02),(0.15±0.04)]。②染料木黄酮组3H-胸腺嘧啶掺入量均显著增加[染料木黄酮组为(101.20±10.06),(844.60±366.90),(512.20±197.6);对照组为(68.67±10.39)],未发现成骨细胞雌激素受体基因和蛋白表达。结论:染料木黄酮不是通过促进雌激素受体基因和蛋白的表达来促进成骨细胞增殖。     

The Eph receptor A4 plays a role in demyelination and depression-related behavior

Proper myelination of axons is crucial for normal sensory, motor, and cognitive function. Abnormal myelination is seen in brain disorders such as major depressive disorder (MDD), but the molecular mechanisms connecting demyelination with the pathobiology remain largely unknown. We observed demyelination and synaptic deficits in mice exposed to either chronic, unpredictable mild stress (CUMS) or LPS, 2 paradigms for inducing depression-like states. Pharmacological restoration of myelination normalized both synaptic deficits and depression-related behaviors. Furthermore, we found increased ephrin A4 receptor (EphA4) expression in the excitatory neurons of mice subjected to CUMS, and shRNA knockdown of EphA4 prevented demyelination and depression-like behaviors. These animal data are consistent with the decrease in myelin basic protein and the increase in EphA4 levels we observed in postmortem brain samples from patients with MDD. Our results provide insights into the etiology of depressive symptoms in some patients and suggest that inhibition of EphA4 or the promotion of myelination could be a promising strategy for treating depression.     

Phosphorylation and desensitization of the human thromboxane receptor-alpha by G protein-coupled receptor kinases

The thromboxane A(2) receptor (TP), which mediates vasoconstriction, mitogenesis, and platelet aggregation, has been shown to undergo rapid agonist-induced desensitization. Two isoforms (alpha and beta) of TP have been recognized. The potential role of the G protein-coupled receptor kinases (GRKs) in the phosphorylation and desensitization of TP alpha was investigated. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged TP alpha was used to study the phosphorylation and desensitization of the receptor. Rapid isolation of the (32)P-labeled receptor was achieved by Ni(2+)-nitrilotriacetic acid agarose after agonist stimulation of HEK293 cells prelabeled with (32)P(i). [1S-[1 alpha,2 alpha(Z),3 beta(1E,3S*),4 alpha]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP) induced receptor phosphorylation and Ca(2+) release in a time- and dose-dependent manner. Pretreatment of cells with I-BOP abolished subsequent induction of Ca(2+) release through a second dose of I-BOP. Transfection with expression plasmids encoding the cDNA of GRK5 or GRK6 augmented I-BOP-induced phosphorylation and inhibited I-BOP-stimulated Ca(2+) release. Both I-BOP-induced and GRK-mediated phosphorylation and phorbol ester-induced phosphorylation were blocked by the addition of 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) (GF 109203X). This indicates that GF 109203X, a known protein kinase C (PKC) inhibitor, also inhibits GRKs. This finding was further supported by in vitro studies in which preparations of GRK5 and GRK6 were found to be inhibited by GF 109203X. These results suggest that GRK5 and GRK6 may phosphorylate the TP alpha in an agonist-dependent manner. Furthermore, the results obtained with PKC inhibitors in assessing the role of PKC in agonist-induced receptor phosphorylation should be interpreted with caution.     

Salt bridges overlapping the gonadotropin-releasing hormone receptor agonist binding site reveal a coincidence detector for G protein-coupled receptor activation

G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function.     

Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion

CagA protein is a major virulence factor of Helicobacter pylori, which is delivered into gastric epithelial cells and elicits growth factor-like responses. Once within the cells, CagA is tyrosine phosphorylated by Src family kinases and targets host proteins required to induce the cell responses. We show that the phosphorylated CagA binds Crk adaptor proteins (Crk-II, Crk-I, and Crk-L) and that the interaction is important for the CagA-mediated host responses during H. pylori infection. H. pylori-induced scattering of gastric epithelial cells in culture was blocked by overexpression of dominant-negative Crk and by RNA interference-mediated knockdown of endogenous Crk. H. pylori infection of the gastric epithelium induced disruption of E-cadherin/catenin-containing adherens junctions, which was also dependent on CagA/Crk signaling. Furthermore, inhibition of the SoS1/H-Ras/Raf1, C3G/Rap1/B-Raf, or Dock180/Rac1/Wiskott-Aldrich syndrome protein family verprolin homologous protein pathway, all of which are involved downstream of Crk adaptors, greatly diminished the CagA-associated host responses. Thus, CagA targeting of Crk plays a central role in inducing the pleiotropic cell responses to H. pylori infection that cause several gastric diseases, including gastric cancer.     

Protease-activated receptors: new concepts in regulation of G protein-coupled receptor signaling and trafficking

Most G protein-coupled receptors (GPCRs) are reversibly activated upon ligand binding. However, activation of protease-activated G protein-coupled receptors (PARs) occurs through an irreversible proteolytic event that results in the generation of a tethered ligand that cannot diffuse away. This unusual mode of PAR activation raises important questions regarding the mechanisms responsible for termination of receptor signaling. There are currently four members of the PAR family. Thrombin activates PAR1, PAR3, and PAR4, whereas multiple trypsin-like serine proteases activate PAR2. The regulation of signaling by PAR1 has been extensively studied, whereas considerably less is known about the other PARs. It has been demonstrated that rapid termination of PAR1 signaling is critical in determining the magnitude and kinetics of the cellular protease response. Therefore, elucidating the molecular mechanisms involved in the regulation of PAR signaling is essential to fully understand protease-mediated responses. Recent findings indicate that novel mechanisms contribute to PAR1 desensitization, internalization, and down-regulation. This review focuses on the intracellular mechanisms that regulate PAR signaling and the recent progress in developing inhibitors of PAR signaling.     

Mast cell-derived angiopoietin-1 plays a critical role in the growth of plasma cell tumors

Multiple myeloma in humans is frequently associated with mast cell infiltration and neovascularization, which correlate directly with disease severity, but the mechanisms underlying this relationship remain unclear. Here, we report that primary murine mast cells express angiopoietin-1 (Ang-1) and low levels of VEGF-A but not Ang-2 and that 2 established murine plasmacytoma cell lines express high levels of VEGF-A but little or no Ang-1 or Ang-2. An in vivo angiogenesis assay using extracellular matrix components shows that mast cells and plasmacytoma cells, together, promote marked neovascularization composed of dilated vessels, which is prevented by neutralization of VEGF-A and Ang-1 but is only partially reduced by neutralization of either VEGF-A or Ang-1. Mast cells within extracellular matrix components express Ang-1, and recombinant Ang-1 together with plasmacytoma cells promotes extracellular matrix neovascularization similar to that induced by mast cells. A transplantation assay shows that primary mast cells accelerate tumor growth by established plasmacytoma cell lines and that neutralization of Ang-1 alone or with VEGF-A reduces significantly the growth of plasmacytomas containing mast cells. These results demonstrate that mast cell-derived Ang-1 promotes the growth of plasmacytomas by stimulating neovascularization and provide further evidence supporting a causal relationship between inflammation and tumor growth.     

Agonist-induced desensitization/resensitization of human G protein-coupled receptor 17: a functional cross-talk between purinergic and cysteinyl-leukotriene ligands

G protein-coupled receptor (GPR) 17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP-glucose) and cysteinyl-leukotrienes (cysLTs, as LTD(4)). By bioinformatic analysis, two distinct binding sites have been hypothesized to be present on GPR17, but little is known on their putative cross-regulation and on GPR17 desensitization/resensitization upon agonist exposure. In this study, we investigated in GPR17-expressing 1321N1 cells the cross-regulation between purinergic- and cysLT-mediated responses and analyzed GPR17 regulation after prolonged agonist exposure. Because GPR17 receptors couple to G(i) proteins and adenylyl cyclase inhibition, both guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding and the cAMP assay have been used to investigate receptor functional activity. UDP-glucose was found to enhance LTD(4) potency in mediating activation of G proteins and vice versa, possibly through an allosteric mechanism. Both UDP-glucose and LTD(4) induced a time- and concentration-dependent GPR17 loss of response (homologous desensitization) with similar kinetics. GPR17 homologous desensitization was accompanied by internalization of receptors inside cells, which occurred in a time-dependent manner with similar kinetics for both agonists. Upon agonist removal, receptor resensitization occurred with the typical kinetics of G protein-coupled receptors. Finally, activation of GPR17 by UDP-glucose (but not vice versa) induced a partial heterologous desensitization of LTD(4)-mediated responses, suggesting that nucleotides have a hierarchy in producing desensitizing signals. These findings suggest a functional cross-talk between purinergic and cysLT ligands at GPR17. Because of the recently suggested key role of GPR17 in brain oligodendrogliogenesis and myelination, this cross-talk may have profound implications in fine-tuning cell responses to demyelinating and inflammatory conditions when these ligands accumulate at lesion sites.