Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).
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PMID:Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system. 1206 55

The structure-activity relationship and signal transduction properties of the pro-opiomelanocortin (POMC)-derived gamma-MSH peptides in the GH3 cell line was compared with that described for the known melanocortin receptors (MCRs). Single alanine replacements showed that, unlike the classical MCRs, the His(5)-Phe(6)-Arg(7)-Trp(8) sequence in gamma2-MSH is not a core sequence for activating the gamma-MSH receptor in GH3 cells, whereas Met(3) is essential. gamma2-MSH increased binding of [35S]GTPgammaS to membrane preparations of GH3 cells. Blockade of protein kinase A abolished the [Ca(2+)](i) responses to gamma3-MSH, and low nanomolar doses of gamma3-MSH increased intracellular cAMP levels, which could be blocked by pertussis toxin (PTX). We conclude that the putative novel gamma-MSH receptor in GH3 cells is a GPCR, but with structure-activity and signal transduction features different from those of the classical MCRs.
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PMID:Structure-activity relationship and signal transduction of gamma-MSH peptides in GH3 cells: further evidence for a new melanocortin receptor. 1212 34

Adhesion molecules on respiratory epithelial cells play a critical role in inflammatory cell recruitment and accumulation at sites of inflammation. Bordetella pertussis colonizes the human respiratory tract by infecting epithelial cells, leading to an inflammatory response. In this study, the role of bacterial factors in the expression of intercellular adhesion molecule-1 (ICAM-1) on human respiratory epithelial cells was investigated in response to B. pertussis. Flow cytometry and real time RT-PCR analysis showed that BEAS-2B human bronchial epithelial cells expressed increased levels of ICAM-1 mRNA and surface protein in response to B. pertussis infection. Filamentous hemagglutinin (FHA) played a role in this response because of the impaired capability of a FHA-deficient isogenic strain. A mutant strain in which an Arg-Gly-Asp (RGD) site of FHA had been changed to Arg-Ala-Asp had diminished ability to up-regulate ICAM-1 expression. RGD sequence-associated up-regulation of ICAM-1 expression was also observed in primary normal human bronchial epithelial cells. Pretreatment of cells with integrin antagonists such as RGD-containing peptide and antibody against very late antigen-5 (VLA-5) inhibited the up-regulation of ICAM-1 expression, suggesting the participation of VLA-5 integrin in this response. Pertussis toxin (PT) prevented the up-regulation of ICAM-1 expression because a PT-deficient mutant strain induced higher levels of ICAM-1 mRNA and surface protein than the parental strain. Consistent with this, purified PT suppressed the up-regulation of epithelial ICAM-1 expression. These findings demonstrate that B. pertussis FHA up-regulates ICAM-1 expression on respiratory epithelial cells through interaction of its RGD site with host cell VLA-5 integrin, and that PT impairs this response.
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PMID:Bordetella pertussis infection of human respiratory epithelial cells up-regulates intercellular adhesion molecule-1 expression: role of filamentous hemagglutinin and pertussis toxin. 1222 Sep 88

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca2+ concentration ([Ca2+]i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27-31, EGSLQ), but not the des (27-31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca2+]i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca2+]i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca2+]i response, and those with Ala at positions 2-5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca2+]i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na-,K+ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca2+]i via G-protein-coupled pathways, giving downstream enzyme effects.
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PMID:Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells. 1222 64

Angiotensin receptors are highly expressed in neonatal spinal cord. To identify their influence on neuronal excitability, we used patch-clamp recordings in spinal cord slices to assess responses of neonatal rat (5-12 days) ventral horn neurons to bath-applied angiotensin II (ANG II; 1 microM). In 14/34 identified motoneurons tested under current clamp, ANG II induced a slowly rising and prolonged membrane depolarization, blockable with Losartan (n = 5) and (Sar(1), Val(5), Ala(8))-ANG II (Saralasin, n = 4) but not PD123319 (1 microM each; n = 4). Under voltage clamp (V(H) -65 mV), 7/22 motoneurons displayed an ANG-II-induced tetrodotoxin-resistant inward current (-128 +/- 31 pA) with a similar time course, an associated reduction in membrane conductance and net current reversal at -98.8 +/- 3.9 mV. Losartan-sensitive ANG II responses were also evoked in 27/78 tested ventral horn "interneurons." By contrast with motoneurons, their ANG-II-induced inward current was smaller (-39.9 +/- 5.2 pA) and analysis of their I-V plots revealed three patterns. In eight cells, membrane conductance decreased with net inward current reversing at -103.8 +/- 4.1 mV. In seven cells, membrane conductance increased with net current reversing at -37.9 +/- 3.6 mV. In 12 cells, I-V lines remained parallel with no reversal within the current range tested. Intracellular dialysis with GTP-gamma-S significantly prolonged the ANG II effect in seven responsive interneurons and GDP-beta-S significantly reduced the ANG II response in four other cells. Peak inward currents were significantly reduced in all 13 responding neurons recorded in slices incubated in pertussis toxin (5 microgram/ml) for 12-18 h or in 12 neurons perfused with N-ethylmaleimide. Of 29 interneurons sensitive to pertussis toxin or N-ethylmaleimide treatment, 9 cells displayed a decrease in membrane conductance that reversed at -101.3 +/- 3.8 mV. In eight cells, membrane conductance increased and reversed at -38.7 +/- 3.4 mV. In 12 cells, the I-V lines remained parallel with no reversal within the current range tested, suggesting that both conductances are modulated by pertussis toxin-sensitive G proteins. These observations reveal a direct, G-protein-mediated depolarizing action of ANG II on neonatal rat ventral horn neurons. They also imply involvement of two distinct conductances that are differentially distributed among different cell types.
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PMID:Angiotensin AT(1)-receptors depolarize neonatal spinal motoneurons and other ventral horn neurons via two different conductances. 1242 18

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.
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PMID:Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase, extracellular signal-regulated kinases, and intracellular calcium pathways. 1252 46

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.
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PMID:Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor. 1282 Nov 36

To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.
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PMID:A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors. 1469 36

The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.
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PMID:Mutation of cysteine 214 in Gi1 alpha subunit abolishes its endogenous GTPase activity. 1472 8

While it has been shown that the angiotensin type-2 (AT(2)) receptor plays an important role in the development and differentiation of many tissues, the second messengers involved in its signaling pathways are just beginning to be understood. To further determine the signaling pathways for the AT(2) receptor, we have investigated whether human angiotensin type-2 receptor transfected into Chinese hamster ovary (CHO) cells can modulate insulin-induced extracellular signal-related protein kinase (ERK-2) phosphorylation via a G-protein coupled mechanism. Our results indicate that the human AT(2) receptor decreases insulin-induced ERK-2 phosphorylation through a G-protein mediated pathway since inhibition was attenuated by pertussis toxin (a G(i)/G(0) inhibitor). Our findings further indicate that the inhibitory response was insensitive to sodium orthovanadate (a PTPase inhibitor), but sensitive (attenuated) to okadaic acid, suggesting an important role for protein phosphatase 2A (PP2A). We have also shown that alanine substitution of the putative G-protein coupling DRY(141-143) motif of the second intracellular loop significantly decreases the human AT(2) receptor's ability to inhibit insulin-induced ERK-2 phosphorylation. Our results support the hypothesis that the AT(2) receptor inhibits insulin-induced ERK-2 activity via a G-protein coupled pathway involving the up-regulation of PP2A.
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PMID:Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway. 1509 86


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