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At least 919 records · Page 51Linked to original sources
PubMed1998

Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T-lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists. Potential role in beta2-adrenoreceptor desensitization.

In this study, a potential mechanism of beta2-adrenoreceptor desensitization has been explored that is based upon the enhanced degradation of cAMP by phosphodiesterase (PDE). Pretreatment of Jurkat T-cells with 8-bromo cAMP (8-Br-cAMP) or prostaglandin E2 increased PDE3 and PDE4 activity in an actinomycin D- and cycloheximide-sensitive manner. This effect was associated with increased expression of HSPDE3B, HSPDE4A4, HSPDE4D1, HSPDE4D2, and HSPDE4D3 mRNA transcripts. Western analysis reproducibly labeled a band of immunoreactivity in vehicle-treated cells that corresponded to HSPDE4A4 (125 kDa). Although the intensity of this band was unchanged in cells treated with 8-Br-cAMP, additional 68-72-kDa proteins (HSPDE4D2, HSPDE4D1) were labeled that were not detected after vehicle. Similar results were obtained with T-lymphocytes exposed to 8-Br-cAMP and fenoterol. However, in those experiments HSPDE4A4 and HSPDE4D1 appeared to be equally expressed in vehicle- and treated cells, whereas HSPDE4D2 (72 kDa) was detected only after 8-Br-cAMP. The up-regulation of PDE activity in Jurkat T-cells abolished the ability of isoproterenol to elevate cAMP, which was partially reversed by the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine, and by the PDE3 and PDE4 inhibitors, Org 9935 and rolipram, respectively. Collectively, these data suggest that chronic treatment of T-cells with cAMP-elevating agents compromises beta2-adrenoreceptor-mediated cAMP accumulation by increasing the expression of HSPDE3B and HSPDE4D gene products.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.

Animals↗
PubMed2000

Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors.

Mechano-sensitive and fatty acid-activated K(+) belong to the structural class of K(+) channel with two pore domains. Here, we report the isolation and the characterization of a novel member of this family. This channel, called TREK2, is closely related to TREK1 (78% of homology). Its gene is located on chromosome 14q31. TREK2 is abundantly expressed in pancreas and kidney and to a lower level in brain, testis, colon, and small intestine. In the central nervous system, TREK2 has a widespread distribution with the highest levels of expression in cerebellum, occipital lobe, putamen, and thalamus. In transfected cells, TREK2 produces rapidly activating and non-inactivating outward rectifier K(+) currents. The single-channel conductance is 100 picosiemens at +40 mV in 150 mm K(+). The currents can be strongly stimulated by polyunsaturated fatty acid such as arachidonic, docosahexaenoic, and linoleic acids and by lysophosphatidylcholine. The channel is also activated by acidification of the intracellular medium. TREK2 is blocked by application of intracellular cAMP. As with TREK1, TREK2 is activated by the volatile general anesthetics chloroform, halothane, and isoflurane and by the neuroprotective agent riluzole. TREK2 can be positively or negatively regulated by a variety of neurotransmitter receptors. Stimulation of the G(s)-coupled receptor 5HT4sR or the G(q)-coupled receptor mGluR1 inhibits channel activity, whereas activation of the G(i)-coupled receptor mGluR2 increases TREK2 currents. These multiple types of regulations suggest that TREK2 plays an important role as a target of neurotransmitter action.

Amino Acid Sequence↗
PubMed2002

A highly effective dominant negative alpha s construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways.

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative alpha(s) mutant containing substitutions that alter distinct functions was produced and tested for its effects on G(s)-coupled receptor activity in HEK-293 cells. Mutations in the alpha3beta5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for betagamma, and A366S, which decreases affinity for GDP. This triple alpha(s) mutant can inhibit signaling to G(s) from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this alpha(s) mutant blocks all signaling from the calcitonin receptor to G(q). These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein alpha subunits can compete with each other for binding to the same receptor. This dominant negative alpha(s) construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.

Cell Line↗
PubMed2002

Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus.

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.

Animals↗
PubMed1998

VIP induces the translocation and degradation of the alpha subunit of Gs protein in rat pituitary GH4C1 cells.

It has been shown that G proteins are potential regulatory molecules in the transmembrane signaling cascade. The aim of this study was to examine the possibility of equivalent G-protein redistribution and/or down-regulation in a target cell upon agonist stimulation. Short-term (0-80 min) incubation of rat pituitary GH4C1 cells with vasoactive intestinal peptide (VIP, 0.1 microM) induced a decrease in the levels of Gsalpha in the membrane fraction, whereas immunoblot analysis and reconstitution assay of adenylyl cyclase clearly showed an increase in the amount of Gsalpha in the supernatant (cytosolic) fraction. The VIP-induced release of G proteins alpha subunits from membranes was specific for Gsalpha. The VIP-dependent release of Gsalpha from membranes was blocked by a VIP-receptor antagonist, (N-Ac-Tyr,D-Phe)-GRF(1-29)-NH2 (10 microM). Pituitary adenylate cyclase-activating polypeptide (PACAP) also stimulated the release of Gsalpha from membranes of GH4C1 cells. Furthermore, prolonged exposure of cells to VIP (0.1 microM) for 2-24 h caused a 21-40% decrease in Gsalpha from membranes and a 6% increase in total Gsalpha in the cytosolic fraction. The effect of VIP was dose-dependent with ED50 values of 81.6+/-20.0 nM for down-regulation and 2.5+/-0.3 nM for translocation of Gsalpha. Concurrent treatment of GH4C1 cells with VIP and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of VIP. Moreover, the chase experiment of 35S-labeled Gsalpha clearly demonstrated a more rapid rate of decay in the cells maintained in the presence of the agonist. These data indicate that VIP-receptor activates Gsalpha protein and induces the release of Gsalpha from membranes along with its down-regulation in cellular levels.

Animals↗
PubMed1997

An activating mutation in a Caenorhabditis elegans Gs protein induces neural degeneration.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal-transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. We characterized a Caenorhabditis elegans G(s) gene, gsa-1, which encodes a G(s) alpha-subunit (G alpha(s)) that is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of G alpha(s) expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for G alpha(s) in the regulation of these behaviors. Expression of a constitutively active form of G alpha(s) from an inducible promotor results in hypercontraction of body-wall muscle cells and vacuolization and degeneration of neurons within hours of induction. Neurons that are susceptible to the degeneration induced by activated G alpha(s) are predominantly motoneurons located within the ventral nerve cord. Phenotypic analysis shows that the induced neural degeneration is not the result of programmed cell death but is probably caused by the activation of ion channels. A genetic suppressor of activated G alpha(s) was isolated that identifies a putative downstream target of G(s) signaling.

Animals↗
PubMed2001

The xenograft antigen in complex with GS-1-B4 lectin: crystallization and preliminary X-ray analysis.

The implantation of animal organs is one approach to overcoming the shortage of human donor organs for medical transplantation. Although readily available, non-primate tissues are subject to hyperacute rejection wherein human anti-Galalpha(1-3)Gal antibodies react with haptens present on the transplanted cells' surfaces. The understanding of this interaction on a molecular level will further the development of a strategy for the prevention of hyperacute rejection in xenotransplantation. The Galalpha(1-3)Gal hapten ('xenograft antigen') has been cocrystallized with the Gal-specific B(4) isolectin of Griffonia simplicifolia lectin-1. Crystals were analyzed by cryocrystallography and were found to diffract to moderately high resolution on a rotating-anode X-ray source. They belong to the P2(1)2(1)2 space group, with unit-cell parameters a = 111.0, b = 51.3, c = 76.9 A, and contain two molecules per asymmetric unit.

Antigens, Heterophile↗
PubMed1988

Variations in guanine-binding proteins (Gs, Gi) in cultured bovine adrenal cells. Consequences on the effects of phorbol ester and angiotensin II on adrenocorticotropin-induced and cholera-toxin-induced cAMP production.

The corticotropin (ACTH) or cholera-toxin-induced cAMP production by cultured bovine adrenal cells increased progressively between days 0 and 7 of culture. Angiotensin II (A-II), which inhibited both basal and ACTH-stimulated adenylate cyclase of crude adrenal membranes, had no effect on ACTH-induced or cholera-toxin-induced cAMP production by fresh isolated cells (day 0) but progressively potentiated the stimulatory action of both effectors from day 0----1 to day 7 of culture. In contrast, phorbol ester had a potentiating effect on fresh isolated cells. Pretreatment of cells with pertussis toxin enhanced the potentiating effect of A-II on cells between 0 and 3 days of culture, but not after 7 days. ADP-ribosylation by cholera toxin (ribosylating alpha s proteins) or pertussis toxin (alpha i proteins), of adrenal membranes prepared from fresh isolated or cultured cells revealed an increase in alpha s and a dramatic decrease in alpha i, the ratios alpha i/alpha s on days 0, 3 and 7 of culture were 4, 0.6 and 0.1 respectively. These results indicate that (a) A-II had a double effect on ACTH-induced or cholera-toxin-induced cAMP production: one inhibitory mediated by Gi, the other stimulatory mediated by protein kinase C activation; this could explain the lack of apparent effect of A-II on fresh cells; (b) the progressive decrease of alpha i might be responsible for the appearance of the potentiating effect of A-II whereas the progressive increase of alpha s could explain the enhanced responsiveness to ACTH or cholera toxin of cultured cells.

Adenosine Diphosphate Ribose↗
PubMed1993

Synergistic interaction of muscarinic and opioid receptors with GS-linked neurotransmitter receptors to stimulate adenylyl cyclase activity of rat olfactory bulb.

We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with l-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal adenylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect 125I-CRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5'-O-(3'-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarinic and opioid effects. Thus, the synergistic interaction appears to result from opioid- and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.

Adenosine Diphosphate Ribose↗
PubMed1990

NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

Adenosine↗