[Novel functions and regulatory mechanisms of G protein-coupled receptors].
Explore the source record for details and available documents.
GTP-Binding Proteins↗
Biomedical subjects
Norimichi Nakahata
Publications and source records attributed to Norimichi Nakahata.
[Role of lipid rafts in trimeric G protein-mediated signal transduction].
Lipid rafts and caveolae are microdomains in the cell membranes, which contain cholesterol, glycolipids, and sphingomyelin. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are considered to be unstable, being dynamically produced and degraded. Recent studies have reported that lipid rafts contain many signaling molecules, such as glycosylphosphatidylinositol-anchored proteins, acylated proteins, G-protein-coupled receptors (GPCRs), trimeric and small G-proteins and their effectors, suggesting that the lipid rafts have an important role in receptor-mediated signal transduction. Therefore drugs that modify the composition of lipid rafts might influence the efficacy of cellular signal transduction. In this review, we demonstrate the role of lipid rafts in GPCR-G-protein signaling and also present our recent results showing that the wasp toxin mastoparan modifies G(q/11)-mediated phospholipase C activation through the interaction with gangliosides in lipid rafts.
Cholesterol↗
[Regulation of G protein-coupled receptor function by its binding proteins].
G protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A(2) receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPalpha and TPbeta. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28gamma and proteasome subunit alpha7, were identified as direct interacting proteins for TPbeta. TPbeta has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPalpha and TPbeta formed heterodimers, and the signal transduction through TPalpha was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.
Animals↗
Distinct effects of z-335, a new thromboxane A2 receptor antagonist, on rabbit platelets and aortic smooth muscle.
The effect of a novel thromboxane A2 receptor (TP) antagonist, (+/-)-sodium[2-(4-chlorophenylsulfonylaminomethyl)- indan-5-yl]acetate monohydrate (Z-335), on the U46619-induced responses was compared between rabbit platelets and aorta. Z-335 inhibited platelet shape change induced by U46619 with higher efficacy than SQ29548, a common TP antagonist. The U46619-induced platelet aggregation was inhibited by Z-335 in a noncompetitive manner, while it was competitively inhibited by SQ29548. Z-335 inhibited U46619-induced vasoconstriction of rabbit aorta with higher efficacy than SQ29548. The pA2 value of Z-335 in aortic vasoconstriction was significantly higher than in platelet shape change. The competitive binding study showed the higher pKi value of Z-335 against [3H]-SQ29548 binding in rabbit aortic smooth muscle cells than in platelets. These data suggest that Z-335 has useful characteristics of TP antagonism.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes.
UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner. PD98059, a MEK (mitogen-activated protein kinase kinase) inhibitor, and BAPTA-AM [O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester], an intracellular Ca(2+) chelator, reduced UTP-induced ERK phosphorylation and IL-6 mRNA expression. 2-APB [(2-aminoethoxy)diphenylborane], an inositol 1,4,5-trisphosphate (IP(3))-receptor antagonist, inhibited UTP-induced IL-6 mRNA expression; and the action of A23187, a Ca(2+) ionophore, resembled the action of UTP. In contrast, protein kinase C (PKC) downregulation and pertussis toxin did not affect UTP-induced IL-6 mRNA expression, suggesting that PKC and G(i) are not involved in the UTP-induced IL-6 production. However, AG1478, an epidermal growth factor (EGF)-receptor inhibitor, partially decreased UTP-induced ERK phosphorylation and IL-6 expression. These results suggest that UTP-induced IL-6 production is in part mediated via phosphorylation of ERK through G(q/11)/IP(3)/[Ca(2+)](i) and transactivation of the EGF receptor.
Calcium Channel Blockers↗
Complanadines C and D, new dimeric alkaloids from Lycopodium complanatum.
Two new dimeric Lycopodium alkaloids, complanadines C (1) and D (2), have been isolated from the club moss Lycopodium complanatum, and the structures and relative stereochemistry of 1 and 2 were elucidated on the basis of the spectral data. Complanadine D (2) enhanced mRNA expression for NGF.
Alkaloids↗
Thromboxane A2 receptor-mediated G12/13-dependent glial morphological change.
Glial cells express thromboxane A(2) receptor, but its physiological role remains unknown. The present study was performed to examine thromboxane A(2) receptor-mediated morphological change in 1321N1 human astrocytoma cells. Thromboxane A(2) receptor agonists U46619 and STA(2) caused a rapid morphological change to spindle shape from stellate form of the cells pretreated with dibutyryl cyclic AMP, but neither carbachol nor histamine caused the change, suggesting that G(q) pathway may not mainly contribute to the change. Rho kinase inhibitor Y-27632 inhibited U46619-induced morphological change, and U46619 increased the GTP-bound form of RhoA accompanied with actin stress fiber formation. These responses were reduced by expression of p115-RGS that inhibits G(12)/(13) signaling pathway. U46619 also caused the phosphorylation of extracellular signal-regulated kinase (ERK) and [(3)H]thymidine incorporation mainly through G(12)/(13)-Rho pathway. These results suggest that stimulation of thromboxane A(2) receptor causes the morphological change with proliferation mainly through G(12)/(13) activation in glial cells.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Lycopladines B-D and lyconadin B, new alkaloids from Lycopodium complanatum.
Four new alkaloids, lycopladines B-D (1-3) and lyconadin B (4), have been isolated from the club moss Lycopodium complanatum and the structures including the stereochemistry were elucidated on the basis of spectral data and modified Mosher's method. Lyconadin B (4) elevated NGF mRNA expression in 1321N1 human astrocytoma cells.
Alkaloids↗
ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.
We evaluated the role of ATP in functions of human HaCaT keratinocytes. ATP was released from HaCaT cells by changing the culture medium. Reverse transcription-polymerase chain reaction analysis revealed that HaCaT cells expressed multiple P2 purinergic receptor mRNAs. UTP was the most potent agonist to increase the intracellular Ca2+ concentration ([Ca2+]i). UTP and ATP caused the accumulation of [3H]inositol phosphates, suggesting that UTP binds to the Gq/11-coupled P2Y receptor. UTP increased IL-6 mRNA and protein levels, and the increases were inhibited by a P2 purinergic receptor antagonist (suramin, 300 microM). While a protein kinase C inhibitor (GF109203X, 10 microM) was without effect, an intracellular free Ca2+ chelator (BAPTA-AM, 50 microM) suppressed UTP-mediated IL-6 induction. These results suggest that 1) ATP is released from HaCaT cells upon physical stimulation and may act as an autocrine molecule, and 2) the stimulation of P2Y receptors causes IL-6 production via mRNA expression through [Ca2+]i elevation.
Adenosine Triphosphate↗
Physiological significance of thromboxane A(2) receptor dimerization.
The thromboxane A(2) receptor (TP), one of the G protein-coupled receptors (GPCRs), consists of two splicing variants, TPalpha and TPbeta, which differ in their C-terminal regions. In the present study, we investigated whether TPalpha and TPbeta formed homo- or hetero-dimers and whether the dimerization changed the function of TP. The immunofluorescent analysis using human embryonic kidney (HEK) 293 cells expressing either FLAG-tagged TPalpha or TPbeta showed that TPalpha is mainly distributed on plasma membranes and TPbeta existed on plasma membranes and within the cells. Co-immunoprecipitation analysis using HEK293 cells expressing both TPalpha and TPbeta showed that TPalpha and TPbeta formed homo- and hetero-dimers. U46619, a TP agonist, caused phosphoinositide hydrolysis and elevation of [Ca(2+)](i) in a concentration-dependent manner in Chinese hamster ovary (CHO) cells expressing TPalpha or TPbeta. The responses were observed to a greater extent in the cells expressing TPalpha than TPbeta. In the cells expressing both TPalpha and TPbeta, U46619-induced responses were observed to a lesser extent than in the cells expressing TPalpha alone. Furthermore, [(3)H]SQ29548 binding showed that the level of the cell surface expression of TP was the following order: the cells expressing TPalpha > TPalpha and TPbeta > TPbeta. These results indicate that TPalpha and TPbeta formed homo- and hetero-dimers, and TP-mediated signaling may be regulated by the hetero-dimer.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Neurotrophic factor production in human astrocytoma cells by 2,5,6-tribromogramine via activation of epsilon isoform of protein kinase C.
It is known that astrocytes secrete several neurotrophic factors to promote the survival of neurons. For the treatment of neuronal disorders, low molecular weight compounds inducing neurotrophic factor synthesis are useful, because neurotrophic factors are polypeptides which cannot cross the blood brain barrier. When rat pheochromocytoma (PC-12) cells were cultivated in the medium of human astrocytoma cells (1321N1) treated with 2,5,6-tribromogramine, they differentiated to neuron-like cells possessing neurites, indicating that 2,5,6-tribromogramine released neurotrophic factors from 1321N1 cells. In fact, 2,5,6-tribromogramine increased nerve growth factor (NGF) protein synthesis and secretion through mRNA expression. 2,5,6-Tribromogramine inhibited carbachol-induced phosphoinositide hydrolysis as well as phorbol 12,13-myristate acetate did. The inhibition was recovered by bisindolylmaleimide I (GF109203X), a specific protein kinase C (PKC) inhibitor, indicating that 2,5,6-tribromogramine may activate PKC. The morphological differentiation of PC-12 cells by the medium treated with 2,5,6-tribromogramine was also reduced by GF109203X. 2,5,6-Tribromogramine translocated PKC-epsilon but not PKC-alpha or PKC-zeta, to membrane fraction from cytosol fraction. These results indicate that 2,5,6-tribromogramine promotes the synthesis and secretion of neurotrophic factors including NGF in 1321N1 cells via an activation of PKC-epsilon.
Animals↗
Neurite outgrowth activity of cyathane diterpenes from Sarcodon cyrneus, cyrneines A and B.
Two new cyathane diterpenes, cyrneines A (1) and B (2), were isolated from the mushroom Sarcodon cyrneus. The structures of the novel diterpenoids were determined by analysis of their spectroscopic data. Neither cyrneine A nor cyrneine B at 100 microM showed cytotoxicity as determined by LDH analysis. The stimulating activity on neurite outgrowth of cyrneines was evaluated. Rat pheochromocytoma cells (PC12), used as a model system of neuronal differentiation, were cultured with cyrneine A (100 microM), cyrneine B (100 microM) or NGF (100 ng/ml) for 24 h. Interestingly, cyrneines A and B significantly promoted neurite outgrowth in addition to NGF as a positive control.
Animals↗
Participation of epoxygenase activation in saikogenin D-induced inhibition of prostaglandin E(2) synthesis.
We examined the effect of saikogenin D on arachidonic acid metabolism in C6 rat glioma cells to clarify its anti-inflammatory mechanism. Incubation of C6 cells with saikogenin D for 20 min resulted in the inhibition of prostaglandin E(2) production and the accumulation of an arachidonic acid metabolite that was found to be 11,12-dihydroxyeicosatrienoic acid, a metabolite of 11,12-epoxyeicosatrienoic acid. C6 cells expressed rat epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which converted arachidonic acid to epoxyeicosatrienoic acids. 11,12-Epoxyeicosatrienoic acid inhibited A23187-induced prostaglandin E(2) production and SKF-525A, an inhibitor of epoxygenase, attenuated the saikogenin D-induced inhibition of prostaglandin E(2) production in C6 cells. Furthermore, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid, but not saikogenin D, inhibited the activity of cyclooxygenase in a cell-free condition. These data suggest that saikogenin D activates epoxygenases that rapidly convert arachidonic acid to epoxyeicosanoids and dihydroxyeicosatrienoic acids, and then the metabolites secondarily inhibit prostaglandin E(2) production.
8,11,14-Eicosatrienoic Acid↗
Different pathways for activation of extracellular signal-regulated kinase through thromboxane A2 receptor isoforms.
Thromboxane A2 receptor (TP) consists of two alternatively spliced isoforms, TPalpha and TPbeta, which differ in their cytoplasmic tails. In the present study, we examined the difference in signal transduction of TPalpha and TPbeta, using stably expressing cells of TPalpha and TPbeta. The cells expressing TPalpha (TPalpha-SC2) and TPbeta (TPbeta-SC15) were selected based on the similar binding sites of [3H]-SQ29548, a TP antagonist. U46619, a TP agonist, elicited phosphoinositide hydrolysis in TPalpha-SC2 and TPbeta-SC15 cells with a similar concentration-dependency. U46619 also caused the phosphorylation of extracellular signal-regulated kinase (ERK1/2) in both TPalpha-SC2 and TPbeta-SC15 cells. While the peak of the phosphorylation of ERK1/2 was observed 5 min after addition of U46619 in TPalpha-SC2 cells, the long lasting phosphorylation up to 60 min was in TPbeta-SC15 cells. U46619-induced phosphorylation of ERK1/2 at 5 min was inhibited by pertussis toxin in both cells, suggesting that G(i) is involved in the phosphorylation mediated via both TP isoforms. Interfering G(12/13) activity by overexpression of p115-RGS reduced U46619-induced ERK1/2 phosphorylation in TPbeta-SC15 cells, but not in TPalpha-SC2 cells. H89, an inhibitor of protein kinase A (PKA), reduced U46619-induced ERK1/2 phosphorylation in TPalpha-SC2 cells, but not in TPbeta-SC15 cells. These results indicate that G(i) may be involved in TP-mediated ERK1/2 phosphorylation in both isoforms. In addition, H89-sensitive kinase and G(12/13) may be involved in TP-mediated ERK1/2 phosphorylation in TPalpha and TPbeta, respectively.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G.
The cell-surface localization of GPCRs (G-protein-coupled receptors) has emerged as one of critical factors of the GPCR-mediated signal transduction. It has been reported that the C-termini of GPCRs contain the sequences for sorting the receptors to cell surface. In the present study, we have searched for proteins that interact with the C-terminus of PTH (parathyroid hormone)/PTH-related protein receptor (PTHR) by using the yeast two-hybrid system, and identified a cytoskeletal protein 4.1G (generaltype 4.1 protein) as an interactant with the C-terminus. Immunohistochemical study revealed that both PTHR and 4.1G were co-localized on plasma membranes, when they were transiently expressed in COS-7 cells. When 4.1G or the C-terminal domain of 4.1G (4.1G-CTD), a dominant-negative form of 4.1G, was co-expressed with PTHR in COS-7 cells, 4.1G, but not 4.1G-CTD, facilitated the cell-surface localization of PTHR, determined by cell-surface biotinylation assay. PTH-(1-34) caused phosphorylation of ERK (extracellular-signal-regulated kinase) 1/2 in PTHR-expressed cells mainly mediated through EGF (epidermal growth factor) receptor. The phosphorylation was enhanced by the expression of 4.1G, but not 4.1G-CTD. PTH-(1-34) elevated [Ca2+]i (intracellular Ca2+ concentration) independent of EGF receptor activation, and the elevation was enhanced by the expression of 4.1G, but not 4.1G-CTD. These data indicate that 4.1G facilitates the cell-surface localization of PTHR through its interaction with the C-terminus of the receptor, resulting in the potentiation of PTHR-mediated signal transduction.
Animals↗
Isolation and synthesis of a new aromatic compound, brefelamide, from dictyostelium cellular slime molds and its inhibitory effect on the proliferation of astrocytoma cells.
[structure: see text] We have explored the diversity of secondary metabolites produced by cellular slime molds to examine the possible use of such cellular slime molds as a resource for novel drug development. A new aromatic amide, brefelamide (1), was isolated from methanol extracts of the fruiting bodies of Dictyostelium brefeldianum and D. giganteum. The structure of 1 was determined by spectral means including EIMS and (1)H and (13)C NMR. The total synthesis of 1 was carried out to confirm the structure and obtain sufficient samples for performing biological evaluation. Interestingly, compound 1 inhibited the cellular proliferation of 1321N1 human astrocytoma cells.
Amides↗
Inhibitory effect of naringin on lipopolysaccharide (LPS)-induced endotoxin shock in mice and nitric oxide production in RAW 264.7 macrophages.
Lipopolysaccharide (LPS) has been known to induce endotoxin shock via production of inflammatory modulators such as tumor necrosis factor alpha (TNF-alpha), or nitric oxide (NO). In this study, we have examined the effect of naringin (NG), one of the flavonoids, on LPS-induced endotoxin shock in mice and NO production in RAW 264.7 macrophages. For intraperitoneal (i.p., 20 mg/kg) injection of LPS at 48 h, the survival rate of mice administered with LPS alone (n=10) or pretreated with NG at 10, 30 and 60 mg/kg (i.p.) group (n=10) was 0% or 10%, 50% and 70%, respectively. NG dose-dependently suppressed LPS-induced production of TNF-alpha. LPS-induced production of NO at 6 h (125.89+/-16.35 microM), as measured by nitrite formation, was significantly reduced by NG at 30 or 60 mg/kg for 49.49+/-4.81 or 27.91+/-1.81 microM (P<0.01 vs. LPS alone), respectively. To further examine the mechanism by which NG suppresses LPS-induced endotoxin shock, we used an in vitro model, RAW 264.7 mouse macrophage cells. NG (1 mM) suppressed LPS (0.01, 0.1 or 1 microg/ml)-induced production of NO and the expression of inflammatory gene products such as inducible NO synthase (iNOS), TNF-alpha, inducible cyclooxygenase (COX-2) and interleukin-6 (IL-6) as determined by RT-PCR assay. NG was found to have blocked the LPS-induced transcriptional activity of NF-kappaB in electrophoretic mobility shift assay and reporter assay. These findings suggest that suppression of the LPS-induced mortality and production of NO by NG is due to inhibition of the activation of NF-kappaB.
Animals↗
Mastoparan changes the cellular localization of Galphaq/11 and Gbeta through its binding to ganglioside in lipid rafts.
Although it is known that mastoparan, a wasp venom toxin, directly activates Gi/o, mastoparan-induced biological responses are not always explained by this mechanism. For instance, we have demonstrated previously that mastoparan suppressed phosphoinositide hydrolysis induced by carbachol in human astrocytoma cells (FEBS Lett 206:91-94, 1990). In the present study, we examined whether mastoparan affected phosphoinositide hydrolysis by interacting with lipid rafts in PC-12 cells. Mastoparan inhibited UTP-induced increase in [Ca2+]i and phosphoinositide hydrolysis in a concentration-dependent manner. UTP-induced phosphoinositide hydrolysis occurred in lipid rafts, because methyl-beta-cyclodextrin, a disrupting regent of lipid rafts, inhibited the hydrolysis. Mastoparan changed the localization of Galphaq/11 and Gbeta together with cholesterol from lipid rafts to nonraft fractions or cytosol. These changes were inhibited by ganglioside mixtures, suggesting that mastoparan interacts with gangliosides in lipid rafts. In fact, ganglioside mixtures and neuraminidase, but not sialic acid, attenuated the inhibitory effect of mastoparan on phosphoinositide hydrolysis. Furthermore, fluorescence intensity of tyrosine residue of [Tyr3]mastoparan was potentiated by ganglioside mixtures, suggesting the direct binding of mastoparan to gangliosides. Mastoparan caused cytotoxicity of PC-12 cells in a concentration-dependent manner, determined by LDH release. The mastoparan-induced cytotoxicity was significantly inhibited by neuraminidase or gangliosides. The order of inhibitory potency of gangliosides was GT1b approximately GD1b > GD1a > GM1 >> GQ1b, but asialo-GM1 and sialic acid were inactive. These results suggest that mastoparan initially binds to gangliosides in lipid rafts and then it inhibits phosphoinositide hydrolysis by changing the localization of Galphaq/11 and Gbeta in lipid rafts.
Animals↗