Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.
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PMID:Purification and G protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes. 894 Jan 9

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and alpha2A adrenergic receptors or overexpression of Gbeta1gamma2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gbeta1gamma2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gbetagamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.
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PMID:Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. 902 Jan 93

Previously, we identified peptides that stimulate phosphoinositide hydrolysis in several leukocyte cell lines from mixtures of random hexapeptide sequences. Moreover, the peptides activate phospholipase C via a pertussis toxin-sensitive G protein-coupled receptor. We now investigate the structure-activity relationship of the peptides with the goal of improving the activity of the peptides, as well as the biologic function of the peptides. Substitution of the L-methionine at the C terminus of peptides with D-methionine markedly increased the effectiveness of the peptides. The half-maximal effective concentrations of MKYMPm-NH2 and WKYMVm-NH2 for stimulation of phosphoinositide hydrolysis in U266 cells were 30 and 0.5 nM, respectively. By BIAcore analysis we confirmed the existence of a receptor for WKYMVm-NH2. Furthermore, the intracellular calcium concentration increase induced by WKYMVm-NH2 was not inhibited by several chemoattractants (FMLP, IL-8, platelet-activating factor, C5a, granulocyte-macrophage CSF, and granulocyte CSF) suggests that WKYMVm-NH2 has a unique cell surface receptor on leukocytes. WKYMVm-NH2 stimulated the phosphoinositide hydrolysis in U937, HL60, and U266 cells, as well as in human neutrophils. Moreover, WKYMVm-NH2 is more effective than FMLP in the production of superoxide in human neutrophils. The data suggest that WKYMVm-NH2 may have the ability to activate the microbicidal functions of human neutrophils.
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PMID:A peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils. 902 31

1. The human EP3 prostaglandin receptor is a seven transmembrane, G protein-coupled receptor that couples to inhibition of adenylyl cyclase. The receptor occurs as at least six isoforms which result from alternative splicing. The isoforms are identical over the first 359 amino acids, comprising the seven transmembrane helices, but differ in the carboxyl terminal tail which ranges in length from 6 to 65 amino acids beyond the common region. 2. We have stably expressed in CHO-K1 cells four of the isoforms (EP3I-EP3IV) and a form of the EP3 receptor (T-359) truncated at the carboxyl-terminal region defined by the alternative splicing site at amino acid number 359. 3. Isoforms EP3I and EP3II showed concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase in CHO-K1 cells by the EP3 receptor agonist, sulprostone. The IC50 calculated for sulprostone inhibition was 0.2 nM for EP3I and 0.15 nM for EP3II. The maximum extent of inhibition was 80% for both isoforms. 4. Isoforms EP3III and EP3IV showed marked constitutive activity, inhibiting forskolin-stimulated adenylyl cyclase in the absence of agonist. EP3IV also displayed some agonist-dependent inhibition whereas EP3III was fully constitutively active. 5. The truncated receptor T-359 was fully constitutively active, inhibiting forskolin-stimulated adenylyl cyclase by about 70% in the absence of agonist, and showed no agonist-dependent inhibition, in agreement with a similar truncation of the mouse EP3 receptor. 6. To confirm that differences in cyclic AMP level between isoforms represent constitutive activity, we treated cells with pertussis toxin for 6 h to abolish Gi function. Pertussis toxin reversed sulprostone-mediated inhibition of cyclic AMP formation in EP3I and EP3II and abolished constitutive activity of EP3III, EP3IV and T-359 so that the level of forskolin-stimulated cyclic AMP produced was the same in all cells and similar to that obtained in mock-transfected cells. In mock-transfected cells, sulprostone had no effect on forskolin-stimulated cyclic AMP formation. 7. For these experiments we chose clones that showed similar expression levels of each isoform, as determined by binding of [3H]-prostaglandin E2 (PGE2) (EP3I, 0.71; EP3II, 1.47; EP3IV, 1.59 pmol mg-1 protein). Mock-transfected cells showed no detectable binding of [3H]-PGE2. In addition, we performed a detailed study of the effects of expression level on constitutive activity. Over a six fold range of expression there was no change in the properties of each isoform with regard to whether it was constitutively active or not. 8. The degree of constitutive activity correlated with the inverse of the length of the C-terminal tail of the isoforms. However, no correlation was found between isoforms from human and mouse: whereas EP3II shows no constitutive activity, its mouse homologue, EP3 gamma, shows almost complete constitutive activity, even though the C-terminal domains of the receptors following the splice site differ in only 7 of 29 amino acids.
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PMID:Constitutive activity of human prostaglandin E receptor EP3 isoforms. 915 43

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. Here, we investigated the functional activity of RGS3 and a truncated form of RGS3 on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses revealed the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) was shown recently to function as a negative regulator of pheromone signaling in yeast (Druey, K. M., Blumer, K. J., Kang, V. R., and Kehrl, J. H. (1996) Nature 379, 742-746). Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibited a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates were reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA showed no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 was impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity.
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PMID:A truncated form of RGS3 negatively regulates G protein-coupled receptor stimulation of adenylyl cyclase and phosphoinositide phospholipase C. 918 81

Thirty hours after the beginning of in vitro maturation, porcine oocytes were microinjected with mRNA coding for the rat muscarinic M1 receptor. They were then incubated for 15 h to allow sufficient time for completing maturation, translation of the mRNA, and insertion of the receptor into the plasma membrane. They were then treated with acetylcholine, the receptor's agonist, and its effect on inducing various activation-related changes was examined. Acetylcholine treatment triggered the release of Ca2+ from internal stores that could be blocked by atropine, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertussis toxin (PT) had no effect on the Ca2+ transients induced by acetylcholine, suggesting that the signal transduction pathway involved a PT-insensitive G protein. Electron microscopy revealed that in the injected oocytes, acetylcholine induced cortical granule exocytosis. The oocytes were released from meiotic arrest as evidenced by the decrease in H1 kinase activity measured in the oocytes during the histone H1 kinase assay. After resuming meiosis they entered interphase: 58.8% of the injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholine incubation without prior injection with the receptor mRNA, did not cause these changes. The results provide further evidence that the components of a G protein-mediated signal transduction pathway exist in porcine oocytes and that the activation of this pathway via an exogenously supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activating signal at sperm-induced oocyte activation requires further examination.
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PMID:Activation of porcine oocytes via an exogenously introduced rat muscarinic M1 receptor. 920 84

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.
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PMID:Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells. 921 88

The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.
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PMID:Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes. 921 10

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5-1 microg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G-induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein-coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 microg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.
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PMID:Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. 927 89

Previous results have shown that pertussis toxin-sensitive Gi proteins are likely to be involved in regulating the emigration of mature thymocytes from the thymus. In this study, a low stringency polymerase chain reaction (PCR) approach was used to identify Gi protein-coupled cell surface receptors expressed in mouse thymocytes. Among the ten G protein-coupled receptor cDNA isolated, the most prevalent cDNA encoded a polypeptide highly homologous to the human leukocyte-expressed seven-transmembrane-domain receptor LESTR, also referred to as HIV entry cofactor, fusin, or CXCR4. Isolation of full-length cDNA revealed that alternative RNA splicing produces transcripts encoding two isoforms of the murine LESTR, differing by the presence of two amino acids in the N-terminal portion of the longer protein. Functional reconstitution of recombinant murine LESTR with recombinant heterotrimeric G proteins in baculovirus-infected insect cells showed that both receptor variants mediate stromal cell-derived factor 1alpha activation of the pertussis toxin-sensitive G protein Gi2. Receptor subtype-specific reverse transcriptase-PCR analysis revealed differential expression of the two receptor mRNA in lymphoid tissues and brain, indicating that distinct functions are mediated by the two receptor isoforms in these tissues. The presence of LESTR mRNA in very early thymocytes as well as in immature (CD4+ CD8+) thymocytes suggests that both CD4 and LESTR are co-expressed and render developing human thymocytes susceptible for HIV entry, which may affect generation of both CD4+ CD8- and CD4- CD8+ mature lineages.
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PMID:Two murine homologues of the human chemokine receptor CXCR4 mediating stromal cell-derived factor 1alpha activation of Gi2 are differentially expressed in vivo. 929 51


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