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)

The turkey beta-adrenergic receptor (beta-AR), the m1 and m2 forms of the human muscarinic cholingeric receptor (MAChR) and several other mutant and wild-type G protein-coupled receptors were produced in insect Sf9 cells by infection with recombinant baculoviruses. Maximal expression for most receptors was 5-30 pmol receptor/mg protein (2-15 nmol/liter culture). The receptors displayed typical ligand binding characteristics. The beta-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The beta-AR stimulated endogenous adenylyl cyclase in response to beta-adrenergic agonists. The beta-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant beta-AR catalyzed the agonist-dependent activation of Gs by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with the same efficiency as did the natural beta-AR. The m2 MAChR efficiently catalyzed GTP gamma S binding to Go and to the recently identified G protein Gz (Gx). The m2 MAChR also catalyzed the activation of Gj,1 and Gj,3 weakly. Activation of these same G proteins by the ml MAChR was much less efficient, consistent with its known selectivity for pertussis toxin-insensitive G proteins ("Gp") that have not yet been isolated. The beta-AR and m2 MAChR were characteristically stimulated by reduction of disulfides. These results demonstrate the general utility of the baculovirus system for production of large quantities of native G protein-coupled receptors.
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PMID:Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. 184 79

The regulation of cellular responsiveness to dopamine via the D2 dopamine receptor was investigated in mouse fibroblast Ltk-cells stably expressing the rat D2-short receptor [Nature (Lond.) 336:783-787 (1988)]. Dopamine inhibited forskolin-stimulated cAMP levels in these cells (half-maximal inhibition at 3.9 +/- 1.1 nM), and the inhibition by dopamine was blocked by D2 antagonists and was pertussis toxin sensitive. Treatment of these cells with the D2 agonist quinpirole (1 microM) resulted in desensitization of dopaminergic inhibition of forskolin-stimulated cAMP accumulation, with a approximately 4-fold decrease in the potency of dopamine after 1 hr of treatment. No significant changes in total cellular D2 receptor concentrations were observed, even after prolonged agonist treatment. At longer time points, basal and forskolin-stimulated cellular cAMP levels were increased in treated cells. The effect of D2 agonist treatment on membrane adenylyl cyclase (EC 4.6.1.1) activity was examined. Basal and forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were increased by quinpirole treatment for 24 hr. This sensitization of adenylyl cyclase was blocked by the presence of a D2 antagonist. Pertussis toxin pretreatment blocked the sensitization of adenylyl cyclase by quinpirole, although pertussis toxin also caused increased adenylyl cyclase activity on its own. Sensitization was not dependent upon dopaminergic inhibition of intracellular cAMP levels, because quinpirole treatment in the presence of membrane-permeable cAMP analogs or 3-isobutyl-1-methylxanthine (an inhibitor of cAMP phosphodiesterase) resulted in greater sensitization of adenylyl cyclase activity than quinpirole treatment alone. These results suggest that, in this model system, responsiveness to dopamine via the D2 receptor is regulated by both desensitization of receptor function and sensitization of the stimulatory adenylyl cyclase pathway.
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PMID:Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization. 184 20

A rat D2L dopamine receptor, a splice variant of the D2 receptor, has recently been cloned. When transfected into and stably expressed in Chinese hamster ovary cells, these receptors mediate the inhibition of both basal and forskolin-stimulated cAMP production, as previously described. We examined what role this receptor might play in the production of the second messenger arachidonic acid. The calcium ionophore A23187 stimulated the release of arachidonic acid, and this release of arachidonic acid was potentiated by dopamine in a concentration-dependent manner. Dopamine alone, however, had no effect on arachidonic acid release. Quinpirole, a D2-selective agonist, augmented A23187-stimulated arachidonic acid release, and sulpiride, a D2-selective antagonist, blocked this augmentation. cAMP analogs and agents that activate adenylyl cyclase were utilized in an attempt to overcome this dopamine effect. Forskolin, prostaglandin E2, dibutyryl-cAMP, 8-(4-chlorophenylthio)-cAMP, and pertussis toxin all had no appreciable effect on either A23187-stimulated arachidonic acid release or the dopamine enhancement. Inhibition of protein kinase C using long term phorbol ester desensitization and pharmacological inhibitors diminished the dopamine potentiation of arachidonic acid release. These results suggest that the D2 receptor may be increasing the release of arachidonic acid by a mechanism involving protein kinase C but independent of the D2 receptor's inhibition of adenylyl cyclase.
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PMID:Transfected D2 dopamine receptors mediate the potentiation of arachidonic acid release in Chinese hamster ovary cells. 184 57

Dopaminergic D2 receptors mediate the effect of dopamine on cellular effector systems by means of guanine nucleotide-binding proteins (G proteins). The major biochemical effect evoked by these receptors is the inhibition of adenylyl cyclase. As a consequence, the activation of D2 receptors lowers the intracellular cAMP level. Two cDNAs, originated by alternative splicing of the same gene, have been isolated: D2A and D2B. They code for two proteins of 444 and 415 amino acids. These proteins display high affinity for selective D2 dopamine ligands. D2A differs from D2B by an insertion of 87 nucleotides in its cDNA, which is located in a region of the protein considered important for the coupling to G proteins. To investigate functional differences between the two dopamine D2 receptor isoforms, we transiently expressed them in cultured cells. To do so we developed an assay to study membrane receptors that are coupled to the adenylyl cyclase. Using this assay, we were able to show that the stimulation of the adenylyl cyclase induced by the activation of the beta 2-adrenergic receptor is inhibited more efficiently by D2B than D2A. The effects elicited by the D2 receptors are mediated by pertussis toxin-sensitive G proteins. Treatment of transfected cells with pertussis toxin abolishes the inhibitory effects in a dose- and receptor isoform-dependent manner. Our results suggest that the two dopamine receptor isoforms are differentially coupled to G proteins.
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PMID:Transcription mediated by a cAMP-responsive promoter element is reduced upon activation of dopamine D2 receptors. 184 44

Membranes from 2 K1735 murine melanoma clones of high invasive capacity show increased amounts of pertussis toxin (PT) substrate when compared to a weakly invasive cellular counterpart. Using a panel of specific G-protein antibodies, we identified Gi alpha 2 as the PT-sensitive G-protein uniquely abundant in highly invasive cells. In addition, RNA hybridization results confirm the immunoblot observations that Gi alpha 2 is present at higher levels in strongly invasive cells. This result suggests that the elevated expression of Gi alpha 2 in highly invasive cells is not entirely due to differences in either translational efficiency or protein degradation but is related to altered RNA transcriptional initiation, processing and/or degradation. ADP-ribosylation of Gi alpha-subunits by PT inhibited the fibronectin, laminin and collagen type-IV-stimulated motility of the 2 highly invasive clones, while PT treatment of cells from a poorly invasive clone resulted in little or no reduction of the fibronectin, laminin or collagen type-IV-stimulated lower motility. Furthermore, PT treatment of highly or poorly invasive K1735 clones does not result in any alteration in cellular cAMP accumulation, suggesting that the PT substrate is not linked with the adenylyl cyclase enzyme complex. The data suggest that a PT-sensitive G-protein, probably Gi alpha 2 regulates second messenger pathways that contribute to elevated motility in highly invasive K1735 cells.
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PMID:The role of G-protein in matrix-mediated motility of highly and poorly invasive melanoma cells. 185 Mar 81

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells. Finally, the technique for purifying the catalytic subunit by SDS-polyacrylamide gel electrophoresis may prove useful in studying the interaction of the adenylyl cyclase with other components produced by the bacteria, as well as the interaction of the enzyme with eukaryotic target cells.
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PMID:Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis. 185 26

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other G alpha-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi-2 alpha and Gs alpha protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G alpha gene family, at least two other G alpha subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G alpha subunits were isolated and characterized. The results indicate that this G alpha subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.
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PMID:Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells. 190 75

The mechanism and site(s) of the defect responsible for desensitization to hormone stimulation of adenylyl cyclase (AC) vary with cell type. Plasma membrane preparations were assayed after treatment of primary cultured dog thyroid cells to determine the role of the TSH receptor, stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi), and catalytic unit in AC desensitization. Exposure of cells to TSH or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused time dependent decreases in TSH-stimulated AC and [125I]TSH binding with approximately 50% decreases seen after 18 h; Bt2cAMP was unable to reproduce the TSH effect. Whereas TSH treatment caused concomitant decreases (approximately 25%) in both cyclase activity and [125I]TSH binding after 2 h, TPA treatment decreased AC activity after 6 h and binding only after 18 h. The protein kinase C inhibitor, H-7, prevented TPA-induced but not TSH-induced effects on AC and hormone binding. Membrane AC activation by cholera toxin or forskolin was not altered by 18 h pretreatment of cells with TSH or TPA, indicating that these agents had no apparent effect on intrinsic functionality of either Gs or the catalytic unit. TSH or TPA pretreatment of cells reduced subsequent toxin-mediated AD[32P]-ribosylation of Gs and Gi in isolated membranes. However, the TSH- and TPA-induced decreases in AD[32P]-ribosylation and desensitization do not appear to be due to endogenous ribosylation of G proteins, since treatment of cells with pertussis toxin, for example, to endogenously ribosylate Gi, both increased TSH-stimulated AC activity and failed to affect the ability of TSH or TPA to desensitize. Thus, in this system, although specific hormone-induced AC desensitization and receptor down-regulation conform to several aspects of classic homologous processes, similar effects are also induced by a nonreceptor (phorbol ester) pathway; desensitization, however, can precede down-regulation, possibly due to receptor-Gs uncoupling.
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PMID:Protein kinase C activation mimics but does not mediate thyrotropin-induced desensitization of adenylyl cyclase in cultured dog thyroid cells. 190 97

Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.
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PMID:Inhibitory GTP-binding regulatory protein Gi3 can couple angiotensin II receptors to inhibition of adenylyl cyclase in hepatocytes. 190 48

During the perinatal period, norepinephrine (NE)-stimulated adenylyl cyclase activity increased in interscapular brown adipose tissue (IBAT) membranes and then declined to adult levels by 23 days postpartum. The developmental patterns of NE- and NaF-stimulated activities were identical, indicating that the developmental increase in transmitter-stimulated activity resulted from the increased interaction of alpha-subunit of guanine nucleotide-binding stimulatory protein of adenylyl cyclase (Gs alpha) with the catalytic subunit (C). This increased Gs alpha-C interaction was the result of an increase in Gs alpha specific activity, as assessed in cyc- reconstitution assays, as well as an increased C activity, as assessed by forskolin-Mn(2+)-stimulated adenylyl cyclase activity. Although adenylyl cyclase activity increased during the perinatal period, total Gs alpha levels significantly declined because of the loss of the small-molecular-mass form of Gs alpha. Thus the ratio of large to small form of Gs alpha increased threefold and might have contributed to the perinatal increase in activity. Gi alpha-like proteins, as assessed by pertussis toxin-catalyzed [32P]ADP ribosylation, declined dramatically after birth. However, this loss of Gi alpha did not contribute to developmental changes in adenylyl cyclase activity because pertussis toxin treatment failed to alter NE-stimulated activity. In contrast to G alpha subunits, there were no changes in membrane levels of G beta subunits.
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PMID:Developmental changes in adenylyl cyclase and GTP binding proteins in brown fat. 190 46


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