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G Protein (g + protein)
Kinds of G Protein Terms modified by G Protein Selected AbstractsIncreased aeroallergen-specific interleukin-4-producing T cells in asthmatic adultsCLINICAL & EXPERIMENTAL ALLERGY, Issue 12 2002P. Pala Summary Background Asthma, atopy and some forms of respiratory syncytial virus (RSV) disease are thought to be caused by T cells making IL-4 (Th2 cells). However, not all patients with similar patterns of clinical disease have the same underlying pathogenesis and the ability to detect immunopathogenic T cells by examination of the peripheral blood remains in doubt. With the prospect of specific immunotherapy for diseases caused by T cell subsets, it is important to determine whether peripheral blood mononuclear cell (PBMC) reactivity can be used to establish the presence of immunopathogenic responses and therefore to predict therapeutic effects. Objective To detect IL-4 and IFN-, production as markers of Th1 and Th2 responses in the peripheral blood of atopic and asthmatic adults. Methods PBMC from 22 adult asthmatics (18 of whom were atopic) and 21 non-asthmatic volunteers (ten of whom were atopic) were stimulated with cat, birch and house dust mite allergens, human rhinovirus, RSV and recombinant chimaeric F/G protein from RSV in vitro. ELISPOT assays were used to enumerate cells producing IL-4 and IFN-,. Results Asthmatics had a sixfold increase in frequencies of IL-4-producing cells to cat and birch allergen (median values: 37 vs. 7 per million PBMC, P < 0.01 and 20 vs. 3 per million PBMC, P < 0.04, respectively) compared to non-asthmatics. By contrast, non-asthmatic atopics showed no specific increase in antigen-specific IL-4 responses and there was no evident correlation between skin prick test reactivity and ELISPOT results. Atopics had significantly more IFN- ,-producing cells specific for FG than nonatopics. while IFN-, and IL-4 responses to other antigens were not significantly different. Conclusion Enhanced IL-4 responses to non-viral aeroallergens are seen in adults with asthma, while enhanced IFN-, responses to viral antigen FG were seen in atopics. In practical terms, ELISPOT assays for specific cytokines may provide a method that could be used to monitor antigen-specific T cell responses in peripheral blood. [source] Deficiency of the ,-Subunit of the Stimulatory G Protein and Severe Extraskeletal Ossification,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2000Mark C. Eddy Abstract Progressive osseous heteroplasia (POH) is a rare disorder characterized by dermal ossification beginning in infancy followed by increasing and extensive bone formation in deep muscle and fascia. We describe two unrelated girls with typical clinical, radiographic, and histological features of POH who also have findings of another uncommon heritable disorder, Albright hereditary osteodystrophy (AHO). One patient has mild brachydactyly but no endocrinopathy, whereas the other manifests brachydactyly, obesity, and target tissue resistance to thyrotropin and parathyroid hormone (PTH). Levels of the ,-subunit of the G protein (Gs,) were reduced in erythrocyte membranes from both girls and a nonsense mutation (Q12X) in exon 1 of the GNAS1 gene was identified in genomic DNA from the mildly affected patient. Features of POH and AHO in two individuals suggest that these conditions share a similar molecular basis and pathogenesis and that isolated severe extraskeletal ossification may be another manifestation of Gs, deficiency. [source] Reduced alpha adrenergic mediated contraction of renal preglomerular blood vessels as a function of gender and agingJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2005John C. Passmore Abstract As human males age, a decline in baroreflex-mediated elevation of blood pressure occurs due, at least in part, to a reduction in alpha-1 adrenergic vasoconstrictor function. Alpha adrenergic constriction is mediated by guanosine triphosphate binding Protein (G Protein) coupled signaling pathways. Alpha-1 A/C, B, and D adrenergic receptor expressions, measured by GeneChip array, are not reduced during aging in renal blood vessels of male or female rats. Alpha-1 A GeneChip expression is greater, at all ages studied, in females than in males. Prazosin binding by alpha-1 adrenergic receptors is greater in young adult female rats than in young adult male rats; however, it is reduced with aging in both male and female rats. G alpha q GeneChip expression declines while expression of adrenergic receptor kinase (GRK2) and tyrosine phosphatases (TyrP) increase with aging in male rats. The declines in alpha-1 adrenergic receptor binding and G alpha q expression and also the increases in GRK2 and TyrP expression likely relate to the age-related decline of vasoconstriction in male rats. The information that the expression of alpha-1 A adrenergic receptors is greater in female rats and (GRK2) expression does not increase during aging could relate to the gender differences in vasoconstrictor function with aging. Gene therapy to ameliorate the age-related decline in renal function could possibly reduce the need for renal dialysis. Signaling pathways such as those reviewed herein may provide an outline of the molecular pathways needed to move toward successful renal gene therapy for aging individuals. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source] G Protein ,3 Subunit Gene Variants and Essential Hypertension in the Northern Chinese Han PopulationANNALS OF HUMAN GENETICS, Issue 4 2005Biao Li Summary Recently a novel C825T polymorphism in the G protein ,3 subunit gene was identified that showed an association with hypertension in a German population; the results of studies in other populations have been inconsistent. To examine the contribution of GNB3 polymorphisms to the development of hypertension in the northern Chinese Han population, we conducted a case-control study consisting of 501 hypertensive cases and 503 controls using the G(-350)A, C825T and C1429T polymorphisms. Genotypes of samples were determined by PCR and restriction digestion. Single locus analysis showed a significant association between G(-350)A and hypertension (P = 0.01) but no association for C825T or C1429T. The three polymorphisms were in tight linkage disequilibrium (D,=,1 for G(-350)A-C825T, D,= 0.92 for C825T-C1429T) and a total of 7 haplotypes were observed in the entire population. Haplotype A-C-C was found to be significantly related to hypertension (P = 0.032) and A-C-C carriers had a more than two-fold higher risk of hypertension than non-carriers, after adjustment for BMI and glucose. In conclusion, our study suggests that G(-350)A is a potential functional polymorphism that may be related to hypertension, whereas the C825T and C1429T polymorphisms are not associated with hypertension in the northern Chinese Han population. [source] Alteration in G Proteins and Prolactin Levels in Pituitary After Ethanol and Estrogen TreatmentALCOHOLISM, Issue 5 2008Kirti Chaturvedi Background:, Chronic administration of ethanol increases plasma prolactin levels and enhances estradiol's mitogenic action on the lactotropes of the pituitary gland. The present study was conducted to determine the changes in the pituitary levels of G proteins during the tumor development following alcohol and ethanol treatments. Methods:, Using ovariectomized Fischer-344 female rats, we have determined ethanol and estradiol actions at 2 and 4 weeks on pituitary weight and pituitary cell contents of prolactin, Gs. Gq11, Gi1, Gi2, and Gi3 proteins. Western blots were employed to measure protein contents. Results:, Ethanol increased basal and estradiol-enhanced wet weight and the prolactin content in the pituitary in a time-dependent manner. Chronic exposure of estradiol increased the levels of Gs protein in the pituitary. Unlike estradiol, ethanol exposure did not show significant effect on the basal level of Gs protein, but moderately increased the estradiol-induced levels of this protein. Estradiol exposure enhanced Gq11 protein levels in the pituitary after 2 and 4 weeks, while ethanol treatment failed to alter these protein levels in the pituitary in control-treated or estradiol-treated ovariectomized rats. In the case of Gi1, estradiol but not ethanol increased the level of this protein at 4 weeks of treatment. However, estradiol and ethanol alone reduced the levels of both Gi2 and Gi3 proteins at 2 and 4 weeks of treatment. Ethanol also significantly reduced the estradiol-induced Gi2 levels at 4 weeks and Gi3 level at 2 and 4 weeks. Conclusions:, These results confirm ethanol's and estradiol's growth-promoting and prolactin stimulating actions on lactotropes of the pituitary and further provide evidence that ethanol and estradiol may control lactotropic cell functions by altering expression of specific group of G proteins in the pituitary. [source] Involvement of G Proteins in the Mycelial Photoresponses of Phycomyces,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2004George Tsolakis ABSTRACT Many responses of the zygomycete fungus Phycomyces blakesleeanus are mediated by blue light, e.g. the stimulation of ,-carotene synthesis (photocarotenogenesis) and the formation of fruiting bodies (photomorphogenesis). Even though both responses have been described in detail genetically and biophysically, the underlying molecular events remain unknown. Applying a pharmacological approach in developing mycelia, we investigated the possible involvement of heterotrimeric G proteins in the blue-light transduction chains of both responses. G protein agonists (guanosine triphosphate analogues, cholera toxin, pertussis toxin) mimicked in darkness the effect of blue light for both responses, except for cholera toxin, which was ineffective in increasing the ,-carotene content of dark-grown mycelia. Experiments combining the two toxins indicated that photocarotenogenesis could involve an inhibitory G protein (Gi) type, whereas photomorphogenesis may depend on a transducin (Gt type)-like heterotrimer. The determination of the carB (phytoene dehydrogenase) and chs1 (chitin synthase 1) gene expression under various conditions of exogenous challenge supports the G protein participation. The fluctuations of the time course measurements of the carB and chs1 transcripts are discussed. [source] Neural tube defects and impaired neural progenitor cell proliferation in G,1 -deficient miceDEVELOPMENTAL DYNAMICS, Issue 4 2010Hiroaki Okae Abstract Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein,coupled receptors (GPCRs), and both G, subunits and tightly associated G,, subunits regulate downstream effector molecules. Compared to G, subunits, the physiological roles of individual G, and G, subunits are poorly understood. In this study, we generated mice deficient in the G,1 gene and found that G,1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% G,1,/, embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, G,1,/, embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist-induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by G,i and G,, signaling, were abnormal in G,1,/, neural progenitor cells. These data indicate that G,1 is required for normal embryonic neurogenesis. Developmental Dynamics 239:1089,1101, 2010. © 2010 Wiley-Liss, Inc. [source] Differential expression of sphingosine-1-phosphate receptors 1-5 in the developing nervous systemDEVELOPMENTAL DYNAMICS, Issue 2 2009H. Meng Abstract Sphingosine-1-phosphate (S1P) binds to G protein,coupled receptors and can regulate a wide range of cellular functions. In a previous study, we isolated two key enzymes in the S1P pathway that were expressed in migrating neural crest cells. To determine if S1P receptors are present in neural crest cells or peripheral nervous system, we examine the expression patterns of S1P receptors (S1pr1-5) in mouse, and s1pr1 and s1pr3 in chick embryos. Here, we present a comprehensive expression analysis of these receptors using in situ hybridizations, which provide spatiotemporal information. We showed that S1pr2 was expressed in migrating cranial neural crest cells and enteric neurons. S1pr1 was prominently expressed in the neuroepithelium whereas S1pr4 and S1pr5 were in neurons at later stages. On the contrary, S1pr3 was predominantly detected in non-neuronal cells within and surrounding neural structures. We also described novel expression sites for S1P receptors in the developing nervous system. Developmental Dynamics 238:487,500, 2009. © 2009 Wiley-Liss, Inc. [source] Expression patterns of the opsin 5,related genes in the developing chicken retinaDEVELOPMENTAL DYNAMICS, Issue 7 2008Sayuri Tomonari Abstract The opsin gene family encodes G protein,coupled seven-transmembrane proteins that bind to a retinaldehyde chromophore for photoreception. It has been reported that opsin 5 is expressed in mammalian neural tissue, but its function has been elusive. As a first step to understand the function for opsin 5 in the developing eye, we searched for chicken opsin 5 -related genes in the genome by a bioinformatic approach and isolated opsin 5 cDNA fragments from the embryonic retina by RT-PCR. We found that there are three opsin 5,related genes, designated cOpn5m (chicken opsin 5, mammalian type), cOpn5L1 (chicken opsin 5 - like 1), and cOpn5L2 (chicken opsin 5 - like 2), in the chicken genome. Quantitative PCR analysis has revealed that cOpn5m is the most abundant in the developing and early posthatching neural retina. In situ hybridization analysis has shown that cOpn5m is specifically expressed in subsets of differentiating ganglion cells and amacrine cells. These results suggest that the mammalian type opsin 5 may contribute to the development of these retinal cells in the chicken. Developmental Dynamics 237:1910,1922, 2008. © 2008 Wiley-Liss, Inc. [source] G,, that interacts with adenylyl cyclase in opioid tolerance originates from a Gs proteinDEVELOPMENTAL NEUROBIOLOGY, Issue 12 2006Hoau-Yan Wang Abstract We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between G,, and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra-low-dose naloxone. In the present work, using striatum from chronic morphine-treated rats, we isotyped the G, within Gs and Go heterotrimers that coupled to MOR and compared these to the G, isotype of the G,, that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its G,, to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra-low-dose opioid antagonist cotreatment, leads to a two-pronged stimulation of adenylyl cyclase utilizing both G, and G,, subunits of the Gs protein novel to this receptor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoAEXPERIMENTAL DERMATOLOGY, Issue 2 2004Julien Laboureau Abstract: Physical forces play an important role in regulating cell functions. We applied mechanical strain to human fibroblasts by magnetic attraction of superparamagnetic arginine-glycine-aspartic acid (RGD)-coated beads. We confirmed that the MAP kinases Erk and p38 are activated by mechanical strain, and went further by demonstrating the activation of Elk-1 by mechanical strain, mainly through a MEK-Erk pathway. Transfection of a dominant negative form of the G protein rac-1 (rac T17N), and inhibition of PI3K, an effector of rac-1, efficiently prevented Elk-1 activation by mechanical forces. Transfection with C3 transferase, known to inhibit rhoA, and inhibition of rock (a downstream effector of rhoA), gave similar results. However, contrary to the active form of rhoA (rho G14V), transfection of the active form of rac-1 (rac G12V) induced Elk activation and mimicked the effects of mechanical strain. These results point out that the two small G proteins rhoA and rac-1 participate in cell sensitivity to mechanical strain and lead to the modulation of the Erk pathway. [source] Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomesFEBS JOURNAL, Issue 21 2002François Devesa Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane ,,helices and small ecto- and endodomains. A His6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N -dodecyl-,- d -maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins. [source] Saccharomyces cerevisiae plasma membrane nutrient sensors and their role in PKA signalingFEMS YEAST RESEARCH, Issue 2 2010Marta Rubio-Texeira Abstract The ability to elicit a fast intracellular signal leading to an adaptive response is crucial for the survival of microorganisms in response to changing environmental conditions. Therefore, in order to sense changes in nutrient availability, the yeast Saccharomyces cerevisiae has evolved three different classes of nutrient-sensing proteins acting at the plasma membrane: G protein-coupled receptors or classical receptor proteins, which detect the presence of certain nutrients and activate signal transduction in association with a G protein; nontransporting transceptors, i.e. nutrient carrier homologues with only a receptor function, previously called nutrient sensors; and transporting transceptors, i.e. active nutrient carriers that combine the functions of a nutrient transporter and receptor. Here, we provide an updated overview of the proteins involved in sensing nutrients for rapid activation of the protein kinase A pathway, which belong to the first and the third category, and we also provide a comparison with the best-known examples of the second category, the nontransporting transceptors, which control the expression of the regular transporters for the nutrient sensed by these proteins. [source] Carbonyl cyanide m -chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H+ -ATPase in the yeast Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 4 2008Michele B.P. Pereira Abstract The plasma membrane H+ -ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H+ -ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H+ -ATPase, suggesting a possible connection between calcium signaling and activation of ATPase. [source] 5-hydroxytryptamine-mediated increase in glutamate uptake by the leech giant glial cellGLIA, Issue 8 2006Ingolf C. Hirth Abstract The clearance of synaptically released glutamate is one of the pivotal functions of glial cells. We have studied the role of 5-hydroxytryptamine (5-HT, 30 ,M), a neurotransmitter and neurohormone in the leech central nervous system with a versatile action spectrum, on the efficacy of glial glutamate uptake. The activity of the glutamate uptake carrier in the giant glial cell in isolated ganglia of Hirudo medicinalis was monitored by measuring the membrane current and the change in the intracellular Na+ concentration (Na+i) as induced by the glutamate carrier substrate D -aspartate (D -asp, 1 mM). 5-HT increased the D -asp-induced current (EC50 at 5 ,M) and rise in Na+i, an effect which was mimicked by the membrane-permeable cyclic nucleotide analogue dibutyryl-cyclic AMP (db-cAMP). The adenylyl cyclase inhibitor SQ 22,536 and the protein kinase A antagonist Rp-cAMP inhibited the effect of 5-HT. Blocking the G protein in the giant glial cell by injecting GDP-,-S suppressed the effect of 5-HT, but not the effect of db-cAMP, on the D -asp-induced current. Our results suggest that 5-HT enhances the glial uptake of glutamate via cAMP- and PKA-mediated pathway. © 2006 Wiley-Liss, Inc. [source] P2Y13 receptor is critical for reverse cholesterol transport,HEPATOLOGY, Issue 4 2010Aurélie C. Fabre A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y13 (purinergic receptor P2Y, G protein,coupled, 13) activation is essential for HDL uptake but the potential of P2Y13 as a target to promote RCT has not been documented. Here, we show that P2Y13 -deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y13 -deficient mice. Furthermore, cangrelor, a partial agonist of P2Y13, stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI,knockoutliver) but had no effect in P2Y13 knockout mice, which indicate that P2Y13 -mediated HDL uptake pathway is independent of SR-BI,mediated HDL selective cholesteryl ester uptake. Conclusion: These results establish P2Y13 as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT. (HEPATOLOGY 2010) [source] Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway,HEPATOLOGY, Issue 5 2010Nitika Arora Gupta Glucagon-like peptide 1 (GLP-1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP-1 functions as an incretin and stimulates glucose-mediated insulin production by pancreatic , cells. In this study, we demonstrate that exendin-4/GLP-1 has a cognate receptor on human hepatocytes and that exendin-4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon-like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP-1. Exendin-4 increased the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1), AKT, and protein kinase C , (PKC-,) in HepG2 and Huh7 cells. Small interfering RNA against GLP-1R abolished the effects on PDK-1 and PKC-,. Treatment with exendin-4 quantitatively reduced triglyceride stores compared with control-treated cells. Conclusion: This is the first report that the G protein,coupled receptor GLP-1R is present on human hepatocytes. Furthermore, it appears that exendin-4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease. (HEPATOLOGY 2010) [source] The G protein,coupled receptor G2A: Involvement in hepatic lipid metabolism and gallstone formation in mice,HEPATOLOGY, Issue 4 2008Laura E. Johnson The G2A receptor is a member of the ovarian cancer G protein,coupled receptor 1 family of stress-inducible G protein,coupled receptors. In this study, we examined the hepatobiliary effects of loss of function of G2A in mice fed either a chow or lithogenic diet. G2A-deficient (G2A,/,) mice fed chow had a 25% reduction in biliary phosphatidylcholine content, reduced hepatic gene expression of the phosphatidylcholine transporter adenosine triphosphate,binding cassette B4, and an 8-fold increase in expression of the nuclear receptor liver X receptor (LXR). Despite the increased expression of LXR, transcription of several LXR target genes was reduced. G2A,/, mice fed a lithogenic diet had rapid gallstone formation, an increased cholesterol saturation index, a 2.5-fold increase in farnesoid X receptor expression, a 5-fold increase in LXR expression, and a 90% reduction in cholesterol 7,-hydroxylase expression in comparison with wild-type mice. There were no changes in gallbladder volume. Conclusion: These data demonstrate that the G2A receptor is important for hepatobiliary bile salt, cholesterol, and phospholipid homeostasis and for the pathogenesis of cholesterol gallstone formation. (HEPATOLOGY 2008;48:1138,1148.) [source] Gq,-linked phospholipase C,1 and phospholipase C, are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascadeINSECT MOLECULAR BIOLOGY, Issue 4 2010J. J. Hull Abstract Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store-operated Ca2+ channels, suggesting the involvement of a receptor-activated phospholipase C (PLC). In this study, we found that PLC inhibitors U73122 and compound 48/80 reduced sex pheromone production and that intracellular levels of 3H-inositol phosphate species increased following PBAN stimulation. In addition, we amplified cDNAs from pheromone glands corresponding to PLC,1, PLC,4, PLC, and two G protein , subunits, Go and Gq. In vivo RNA interference-mediated knockdown analyses revealed that BmPLC,1, BmGq1, and unexpectedly, BmPLC,, are part of the PBAN signal transduction cascade. [source] Spatial distribution and differential expression of the PBAN receptor in tissues of adult Helicoverpa spp. (Lepidoptera: Noctuidae)INSECT MOLECULAR BIOLOGY, Issue 3 2007A. Rafaeli Abstract Pheromone-biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone production in many female moths. PBAN-like peptides, with common FXPRLamide C-terminals are found in other insect groups where they have other functions. The ubiquity and multifunctional nature of the pyrokinin/PBAN family of peptides suggests that the PBAN receptor proteins could also be present in a variety of insect tissues with alternative functions from that of sex pheromone biosynthesis. Previously we showed the presence of the PBAN-R in Helicoverpa armigera at the protein level. In the present study we confirm the similarities between the two Helicoverpa species: armigera and zea by (1) demonstrating the presence of the receptor protein in Sf9 cells, cloned to express the HezPBAN receptor, as compared with the endogenous receptor protein, previously shown in H. armigera pheromone glands, and (2) by identifying the nucleotide sequence of the PBAN-R from mRNA of H. armigera pheromone glands. Sequences of the two Helicoverpa spp. are 98% identical with most changes taking place in the 3,-end. We demonstrate the spatial distribution of the PBAN receptor protein in membranes of H. armigera brain (Br), thoracic ganglion (TG) and ventral nerve cord (VNC). We also demonstrate the presence and differential expression of the PBAN receptor gene (using reverse transcription,polymerase chain reaction and reverse transcription,quantitative real-time polymerase chain reaction, respectively) in the neural tissues (Br, TG and VNC) of adult H. armigera female moths as compared with its presence in pheromone glands. Surprisingly, the gene for the PBAN receptor is also detected in the male tissue homologous to the female pheromone gland, the aedeagus, although the protein is undetectable and PBAN does not induce physiological (pheromone production) or cellular (cyclic-adenosine monophosphate production) responses in this tissue. Our findings indicate that PBAN or PBAN-like receptors are present in the neural tissues and may represent a neurotransmitter-like function for PBAN-like peptides. In addition, the surprising discovery of the presence of the gene encoding the PBAN receptor in the male homologous tissue, but its absence at the protein level, launches opportunities for studying molecular regulation pathways and the evolution of these G protein coupled receptors (GPCRs). [source] Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2008Alberto Ferlin Abstract Introduction: Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein,coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency. Materials and Methods: Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27,41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Real-time cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2- deficient mice were studied by static and dynamic histomorphometry and ,CT, respectively. Results: Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose- and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor,ligand complex. Consistent with the human phenotype, bone histomorphometric and ,CT analyses of Rxfp2,/, mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates. Conclusions: This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis. [source] Gs, Mutations in Fibrous Dysplasia and McCune-Albright Syndrome,JOURNAL OF BONE AND MINERAL RESEARCH, Issue S2 2006Lee S Weinstein Abstract Fibrous dysplasia (FD) is a focal bone lesion composed of immature mesenchymal osteoblastic precursor cells. Some FD patients also have hyperpigmented skin lesions (café-au-lait spots), gonadotropin-independent sexual precocity, and/or other endocrine and nonendocrine manifestations (McCune-Albright syndrome [MAS]). MAS results from somatic mutations occurring during early development, resulting in a widespread mosaic of normal and mutant-bearing cells, which predicts that the clinical presentation of each patient is determined by the extent and distribution of abnormal cells. These mutations encode constitutively active forms of Gs,, the ubiquitously expressed G protein ,-subunit that couples hormone receptors to intracellular cAMP generation. These mutations lead to substitution of amino acid residues that are critical for the intrinsic GTPase activity that is normally required to deactivate the G protein. This leads to prolonged activation of Gs, and its downstream effectors even with minimal receptor activation. This explains why MAS patients have stimulation of multiple peripheral endocrine glands in the absence of circulating stimulatory pituitary hormones and increased skin pigment, which is normally induced by melanocyte-stimulating hormone through Gs,/cAMP. Similar mutations are also present in 40% of pituitary tumors in acromegaly patients and less commonly in other endocrine tumors. FD results from increased cAMP in bone marrow stromal cells, leading to increased proliferation and abnormal differentiation. Parental origin of the mutated allele may also affect the clinical presentation, because Gs, is imprinted and expressed only from the maternal allele in some tissues (e.g., pituitary somatotrophs). [source] GNAS1 Mutation and Cbfa1 Misexpression in a Child with Severe Congenital Platelike Osteoma Cutis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2000George L. Yeh Abstract We evaluated a 7-year-old girl with severe platelike osteoma cutis (POC), a variant of progressive osseous heteroplasia (POH). The child had congenital heterotopic ossification of dermis and subcutaneous fat that progressed to involve deep skeletal muscles of the face, scalp, and eyes. Although involvement of skeletal muscle is a prominent feature of POH, heterotopic ossification has not been observed in the head, face, or extraocular muscles. The cutaneous ossification in this patient was suggestive of Albright hereditary osteodystrophy (AHO); however, none of the other characteristic features of AHO were expressed. Inactivating mutations of the GNAS1 gene, which encodes the ,-subunit of the stimulatory G protein of adenylyl cyclase, is the cause of AHO. Mutational analysis of GNAS1 using genomic DNA of peripheral blood and of lesional and nonlesional tissue from our patient revealed a heterozygous 4-base pair (bp) deletion in exon 7, identical to mutations that have been found in some AHO patients. This 4-bp deletion in GNAS1 predicts a protein reading frameshift leading to 13 incorrect amino acids followed by a premature stop codon. To investigate pathways of osteogenesis by which GNAS1 may mediate its effects, we examined the expression of the obligate osteogenic transcription factor Cbfa1/RUNX2 in lesional and uninvolved dermal fibroblasts from our patient and discovered expression of bone-specific Cbfa1 messenger RNA (mRNA) in both cell types. These findings document severe heterotopic ossification in the absence of AHO features caused by an inactivating GNAS1 mutation and establish the GNAS1 gene as the leading candidate gene for POH. [source] Deficiency of the ,-Subunit of the Stimulatory G Protein and Severe Extraskeletal Ossification,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2000Mark C. Eddy Abstract Progressive osseous heteroplasia (POH) is a rare disorder characterized by dermal ossification beginning in infancy followed by increasing and extensive bone formation in deep muscle and fascia. We describe two unrelated girls with typical clinical, radiographic, and histological features of POH who also have findings of another uncommon heritable disorder, Albright hereditary osteodystrophy (AHO). One patient has mild brachydactyly but no endocrinopathy, whereas the other manifests brachydactyly, obesity, and target tissue resistance to thyrotropin and parathyroid hormone (PTH). Levels of the ,-subunit of the G protein (Gs,) were reduced in erythrocyte membranes from both girls and a nonsense mutation (Q12X) in exon 1 of the GNAS1 gene was identified in genomic DNA from the mildly affected patient. Features of POH and AHO in two individuals suggest that these conditions share a similar molecular basis and pathogenesis and that isolated severe extraskeletal ossification may be another manifestation of Gs, deficiency. [source] Adrenergic regulation of cardiac myocyte apoptosisJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2001Krishna Singh The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via ,- and ,-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of ,1 -AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of ,2 -AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of ,1 -AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either ,1 -AR or G,s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of ,2 -AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype ,1B -AR does not result in apoptosis, whereas overexpression of G,q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways. © 2001 Wiley-Liss, Inc. [source] Genetic heterogeneity of G and F protein genes from Argentinean human metapneumovirus strainsJOURNAL OF MEDICAL VIROLOGY, Issue 5 2006Monica Galiano Abstract Human metapneumovirus (hMPV) is a newly identified paramixovirus, associated with respiratory illnesses in all age groups. Two genetic groups of hMPV have been described. The nucleotide sequences of the G and F genes from 11 Argentinean hMPV strains (1998,2003) were determined by RT-PCR and direct sequencing. Phylogenetic analysis showed that hMPV strains clustered into two main genetic lineages, A and B. Strains clustered into A group were split into two sublineages, A1 and A2. All strains belonging to group B clustered with representative strains from sublineage B1. No Argentinean strains belonged to sublineage B2. F sequences showed high percentage identities at nucleotide and amino acid levels. In contrast, G sequences showed high diversity between A and B groups. Most changes observed in the deduced G protein sequence were amino acid substitutions in the extracellular domain, and changes in stop codon usage leading to different lengths in the G proteins. High content of serine and threonine residues were also shown, suggesting that this protein would be highly glycosylated. The potential sites for N- and O-glycosylation seem to have a different conservation pattern between the two main groups. This is the first report on the genetic variability of the G and F protein genes of hMPV strains in South America. Two main genetic groups and at least three subgroups were revealed among Argentinean hMPV strains. The F protein seems to be highly conserved, whereas the G protein showed extensive diversity between groups A and B. J. Med. Virol. 78:631,637, 2006. © 2006 Wiley-Liss, Inc. [source] A phylogenetic study of human respiratory syncytial viruses group A and B strains isolated in two cities in Japan from 1980,2002JOURNAL OF MEDICAL VIROLOGY, Issue 2 2005Yuki Kuroiwa Abstract The circulation pattern and genetic evolution of respiratory syncytial virus (RSV) in Japan were examined based on 109 RSV field strains isolated over 20 seasons (1980,2002) in two cities, Sapporo and Tokyo. The second hypervariable region of the large glycoprotein (G) gene was amplified by RT-PCR and the products sequenced directly. The nucleotide sequences were compared to those representatives of RSV genotypes identified previously. Japanese group A and B isolates clustered into five and four genotypes defined previously, respectively. Another one group A and one group B genotypes, which could not be assigned to previous genotypes, were also identified. Although different genotypes usually co-circulated in each season, the isolates in proximate seasons from two communities were usually located in the same branches. Moreover, the strains with genotypes defined previously were usually isolated at the same time as each reference strain of Western countries. Several mutant group B strains with 1,20 longer amino acid G proteins were newly identified in Sapporo. These findings suggest that Japanese RSV strains underwent geographical and also temporal clustering while participating in RSV genetic evolution in a global setting. In addition, Japanese strains, especially group B, might have evolved individually in each community, sometimes changing the length of the G protein. J. Med. Virol. 76:241,247, 2005. © 2005 Wiley-Liss, Inc. [source] RGS9-2 mediates specific inhibition of agonist-induced internalization of D2 -dopamine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Jeremy Celver J. Neurochem. (2010) 114, 739,749. Abstract Regulator of G protein signaling 9-2 (RGS9-2), a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, a brain region involved in controlling movement, motivation, mood and addiction. RGS9-2 can be found co-localized with D2 -class dopamine receptors in medium spiny striatal neurons and altered functioning of both RGS9-2 and D2 -like dopamine receptors have been implicated in schizophrenia, movement disorders and reward responses. Previously we showed that RGS9-2 can specifically co-localize with D2 -dopamine receptors (D2R). Here we provide further evidence of the specificity of RGS9-2 for regulating D2R cellular functions: the expression of RGS9-2 inhibits dopamine-mediated cellular internalization of D2R, while the expression of another RGS protein, RGS4, had no effect. In addition, the agonist-mediated internalization of the G protein coupled delta opioid receptor was unaffected by RGS9-2 expression. We utilized mutant constructs of RGS9-2 to show that the RGS9-2 DEP (for Disheveled, EGL-10, Pleckstrin homology) domain and the GTPase accelerating activity of RGS9-2 were necessary for mediating specific inhibition of D2R internalization. [source] Amino acids involved in conformational dynamics and G protein coupling of an odorant receptor: targeting gain-of-function mutationJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Aya Kato Abstract Thousands of different odorants are recognized and discriminated by odorant receptors (ORs) in the guanine nucleotide-binding protein (G protein)-coupled seven-transmembrane receptor family. Odorant-bound ORs stimulate Gs-type G proteins, G,olf, which in turn activates cAMP-mediated signaling pathway in olfactory sensory neurons. To better understand the molecular basis for OR activation and G protein coupling, we analyzed the effects of a series of site-directed mutations of mouse ORs, on function. Mutations of conserved amino acid residues in an intracellular loop or the C-terminus resulted in loss of activity without impairing ligand-binding activity, indicating that these residues are involved in G,s/olf coupling. Moreover, mutation of the serine in KAFSTC, the OR-specific sequence motif, resulted in a dramatic increase in odorant responsiveness, suggesting that the motif is involved in a conformational change of the receptor that regulates G protein coupling efficiency. Our results provide insights into how ORs switch from an inactive to an active state, as well as where and how activated ORs interact with G proteins. [source] A family of octapamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogasterJOURNAL OF NEUROCHEMISTRY, Issue 2 2005Sabine Balfanz Abstract In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, ,fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca2+ release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca2+ concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC50 of ,3 × 10,8 m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at ,,100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT,PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors. [source] | |||||||||||||||||||||||||||||||||||||