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1.
J Biol Chem ; 293(10): 3546-3561, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317494

RESUMO

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel-PIP2 interactions. H2S regulation was attenuated when channel-PIP2 interactions were strengthened and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Sulfeto de Hidrogênio/farmacologia , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Sulfetos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Células CHO , Cricetulus , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Sulfeto de Hidrogênio/química , Sulfeto de Hidrogênio/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/química , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfetos/química , Sulfetos/metabolismo , Xenopus laevis
2.
ACS Chem Neurosci ; 7(9): 1292-9, 2016 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-27385190

RESUMO

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.


Assuntos
Potenciais da Membrana/efeitos dos fármacos , Antagonistas do Receptor 5-HT2 de Serotonina/síntese química , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , Bário/farmacologia , Cálcio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Ketanserina/farmacocinética , Ketanserina/farmacologia , Potenciais da Membrana/genética , Mutação/genética , Oócitos , Ligação Proteica/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Risperidona/farmacologia , Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas da Serotonina/farmacocinética , Antagonistas da Serotonina/farmacologia , Trítio/farmacocinética , Xenopus laevis
3.
Sci Signal ; 9(410): ra5, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26758213

RESUMO

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.


Assuntos
Multimerização Proteica , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/metabolismo , Transdução de Sinais , Regulação Alostérica , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Receptor 5-HT2A de Serotonina/genética , Receptores de Glutamato Metabotrópico/genética , Esquizofrenia/genética
4.
Pflugers Arch ; 468(5): 775-93, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26780666

RESUMO

We previously reported that co-expression of the Gi-coupled metabotropic glutamate receptor 2 (mGlu2R) and the Gq-coupled serotonin (5-HT) 2A receptor (2AR) in Xenopus oocytes (Fribourg et al. Cell 147:1011-1023, 2011) results in inverse cross-signaling, where for either receptor, strong agonists suppress and inverse agonists potentiate the signaling of the partner receptor. Importantly, through this cross-signaling, the mGlu2R/2AR heteromer integrates the actions of psychedelic and antipsychotic drugs. To investigate whether mGlu2R and 2AR can cross-signal in mammalian cells, we stably co-expressed them in HEK293 cells along with the GIRK1/GIRK4 channel, a reporter of Gi and Gq signaling activity. Crosstalk-positive clones were identified by Fura-2 calcium imaging, based on potentiation of 5-HT-induced Ca(2+) responses by the inverse mGlu2/3R agonist LY341495. Cross-signaling from both sides of the complex was confirmed in representative clones by using the GIRK channel reporter, both in whole-cell patch-clamp and in fluorescence assays using potentiometric dyes, and further established by competition binding assays. Notably, only 25-30 % of the clones were crosstalk-positive. The crosstalk-positive phenotype correlated with (a) increased colocalization of the two receptors at the cell surface, (b) lower density of mGlu2R binding sites and higher density of 2AR binding sites in total membrane preparations, and (c) higher ratios of mGlu2R/2AR normalized surface protein expression. Consistent with our results in Xenopus oocytes, a combination of ligands targeting both receptors could elicit functional crosstalk in a crosstalk-negative clone. Crosstalk-positive clones can be used in high-throughput assays for identification of antipsychotic drugs targeting this receptor heterocomplex.


Assuntos
Sinalização do Cálcio , Multimerização Proteica , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Receptor 5-HT2A de Serotonina/genética , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/genética
5.
Biochim Biophys Acta ; 1848(10 Pt A): 2406-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26196595

RESUMO

Cholesterol is one of the major lipid components of membranes in mammalian cells. In recent years, cholesterol has emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels in general and on inwardly rectifying potassium (Kir) channels in particular is a decrease in activity. In contrast, we have recently shown that native G-protein gated Kir (GIRK or Kir3) channels that underlie atrial KACh currents are up-regulated by cholesterol. Here we unveil the biophysical basis of cholesterol-induced increase in KACh activity. Using planar lipid bilayers we show that cholesterol significantly enhances the channel open frequency of the Kir3.1/Kir3.4 channels, which underlie KACh currents. In contrast, our data indicate that cholesterol does not affect their unitary conductance. Furthermore, using fluorescent and TIRF microscopy as well as surface protein biotinylation, we also show that cholesterol enrichment in vitro has no effect on surface expression of GFP-tagged channels expressed in Xenopus oocytes or transfected into HEK293 cells. Together, these data demonstrate for the first time that cholesterol enhances Kir3-mediated current by increasing the channel open probability.


Assuntos
Colesterol/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Modelos Estatísticos , Potássio/metabolismo , Animais , Simulação por Computador , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Células HEK293 , Humanos , Modelos Químicos , Oócitos/química , Oócitos/fisiologia , Xenopus laevis
6.
Annu Rev Physiol ; 77: 81-104, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25293526

RESUMO

Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca(2+)]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states.


Assuntos
Canais Iônicos/fisiologia , Proteínas de Membrana/fisiologia , Fosfatidilinositóis/fisiologia , Animais , Membrana Celular/fisiologia , Humanos , Fosforilação/fisiologia , Transdução de Sinais/fisiologia
7.
Cell ; 147(5): 1011-23, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22118459

RESUMO

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.


Assuntos
Antipsicóticos/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais , Anfetaminas/farmacologia , Animais , Clozapina/farmacologia , Dimerização , Relação Dose-Resposta a Droga , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Metisergida/farmacologia , Camundongos , Oócitos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Xenopus
8.
Pflugers Arch ; 461(3): 387-97, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21107857

RESUMO

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2) or PIP(2)] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP(2) and other phosphoinositides. Here, we report a novel requirement for PIP(2) in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP(2) levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP(2) levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP(2) and PIP(2)-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP(2). Neutralization of positively charged amino acids abolished EGFR/PIP(2) interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP(2) and point to the basic JD's critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP(2) may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.


Assuntos
Receptores ErbB/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiologia , Regulação para Baixo , Receptores ErbB/química , Células HeLa , Humanos , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Estrutura Terciária de Proteína
9.
J Biol Chem ; 285(51): 39790-800, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20937804

RESUMO

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the ßγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gßγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.


Assuntos
Proteínas de Bactérias/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Proteínas Recombinantes de Fusão/química , Animais , Proteínas de Bactérias/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Xenopus laevis
10.
J Mol Cell Cardiol ; 49(5): 746-52, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20736017

RESUMO

HIV protease inhibitors (HIV PI) reduce morbidity and mortality of HIV infection but cause multiple untoward effects. Because certain HIV PI evoke production of reactive oxygen species (ROS) and volume-sensitive Cl(-) current (I(Cl,swell)) is activated by ROS, we tested whether HIV PI stimulate I(Cl,swell) in ventricular myocytes. Ritonavir and lopinavir elicited outwardly rectifying Cl(-) currents under isosmotic conditions that were abolished by the selective I(Cl,swell)-blocker DCPIB. In contrast, amprenavir, nelfinavir, and raltegravir, an integrase inhibitor, did not modulate I(Cl,swell) acutely. Ritonavir also reduced action potential duration, but amprenavir did not. I(Cl,swell) activation was attributed to ROS because ebselen, an H(2)O(2) scavenger, suppressed ritonavir- and lopinavir-induced I(Cl,swell). Major ROS sources in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin failed to block ritonavir- or lopinavir-induced currents, although it blocks I(Cl,swell) elicited by osmotic swelling or stretch. In contrast, rotenone, a mitochondrial e(-) transport inhibitor, suppressed both ritonavir- and lopinavir-induced I(Cl,swell). ROS production was measured in HL-1 cardiomyocytes with C-H(2)DCFDA-AM and mitochondrial membrane potential (ΔΨ(m)) with JC-1. Flow cytometry confirmed that ritonavir and lopinavir but not amprenavir, nelfinavir, or raltegravir augmented ROS production, and HIV PI-induced ROS production was suppressed by rotenone but not NADPH oxidase blockade. Moreover, ritonavir, but not amprenavir, depolarized ΔΨ(m). These data suggest ritonavir and lopinavir activated I(Cl,swell) via mitochondrial ROS production that was independent of NADPH oxidase. ROS-dependent modulation of I(Cl,swell) and other ion channels by HIV PI may contribute to some of their actions in heart and perhaps other tissues.


Assuntos
Canais de Cloreto/metabolismo , Inibidores da Protease de HIV/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Carbamatos/farmacologia , Furanos , Inibidores de Integrase de HIV/farmacologia , Lopinavir , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nelfinavir/farmacologia , Pirimidinonas/farmacologia , Pirrolidinonas/farmacologia , Coelhos , Raltegravir Potássico , Ritonavir/farmacologia , Sulfonamidas/farmacologia , Fatores de Tempo
11.
Cardiovasc Res ; 88(1): 93-100, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20444986

RESUMO

AIMS: We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. METHODS AND RESULTS: ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. CONCLUSION: ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.


Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Endotelina-1/metabolismo , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Angiotensina II/metabolismo , Animais , Tamanho Celular , Canais de Cloreto/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Citometria de Fluxo , Técnicas In Vitro , Potenciais da Membrana , Moduladores de Transporte de Membrana/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Pressão Osmótica , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Coelhos , Receptor de Endotelina A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Desacopladores/farmacologia
12.
J Neurosci ; 28(2): 483-90, 2008 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18184791

RESUMO

The role of presenilin-1 (PS1) in neuronal phosphatidylinositol 3-kinase (PI3K)/Akt signaling was investigated in primary neuronal cultures from wild-type (WT) and PS1 null (PS1-/-) embryonic mouse brains. Here we show that in PS1-/- cultures, the onset of neuronal maturation coincides with a decrease in the PI3K-dependent phosphorylation-activation of Akt and phosphorylation-inactivation of glycogen synthase kinase-3 (GSK-3). Mature PS1-/- neurons show increased activation of apoptotic caspase-3 and progressive degeneration preceded by dendritic retraction. Expression of exogenous WT PS1 or constitutively active Akt in PS1-/- neurons stimulates PI3K signaling and suppresses both caspase-3 activity and dendrite retraction. The survival effects of PS1 are sensitive to inhibitors of PI3K kinase but insensitive to gamma-secretase inhibitors. Familial Alzheimer disease (FAD) mutations suppress the ability of PS1 to promote PI3K/AKT signaling, prevent phosphorylation/inactivation of GSK-3 and promote activation of caspase-3. These mutation effects are reversed upon coexpression of constitutively active Akt. Together, our data indicate that the neuroprotective role of PS1 depends on its ability to activate the PI3K/Akt signaling pathway and that PS1 FAD mutations increase GSK-3 activity and promote neuronal apoptosis by inhibiting the function of PS1 in this pathway. These observations suggest that stimulation of PI3K/Akt signaling may be beneficial to FAD patients.


Assuntos
Mutação/fisiologia , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Presenilina-1/genética , Transdução de Sinais/fisiologia , Doença de Alzheimer/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Encéfalo/citologia , Caspases/metabolismo , Células Cultivadas , Cromonas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Neurônios/fisiologia , Transdução de Sinais/efeitos dos fármacos , Simplexvirus/fisiologia
13.
J Neurochem ; 101(3): 674-81, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17254019

RESUMO

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação/fisiologia , Neurotoxinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Presenilina-1/genética , Peptídeos beta-Amiloides/metabolismo , Análise de Variância , Animais , Linhagem Celular , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos de Peptídeos/metabolismo , Presenilina-1/fisiologia , Transfecção
14.
EMBO J ; 25(6): 1242-52, 2006 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-16511561

RESUMO

Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/gamma-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by gamma-secretase. These data show that the PS1/gamma-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, gamma-secretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and gamma-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.


Assuntos
Efrina-B2/metabolismo , Proteínas de Membrana/fisiologia , Metaloproteases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor EphB2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Tirosina Quinase CSK , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Dominantes , Humanos , Rim/citologia , Rim/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Presenilina-1 , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Quinases da Família src
15.
J Biol Chem ; 280(43): 36007-12, 2005 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-16126725

RESUMO

Presenilin1 (PS1), a protein involved in cellular development, forms functional complexes with beta-catenin, a regulator of Wnt signaling and cell-cell adhesion. In addition, both proteins have been shown to play important roles in disease including cancer and Alzheimer disease. Although PS1 and beta-catenin are found in the same complexes, it is not clear whether they bind directly to each other or a third complex component, like cadherin, may mediate their interactions. Here we show that PS1 and beta-catenin form no detectable complexes in cells that express no cadherin. In contrast, these complexes are readily found in E-cadherin containing cells. Furthermore, binding of both PS1 and beta-catenin to E-cadherin is necessary for the formation of PS1/beta-catenin complexes. Importantly, our data show that binding of PS1 to cadherin mediates the effects of PS1 on the phosphorylation, ubiquitination, and destabilization of beta-catenin. Thus, cadherins mediate both the association of PS1 and beta-catenin and the effects of PS1 on the cellular levels of beta-catenin.


Assuntos
Caderinas/metabolismo , Proteínas de Membrana/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Western Blotting , Adesão Celular , Linhagem Celular Tumoral , Detergentes/farmacologia , Humanos , Imunoprecipitação , Substâncias Macromoleculares/metabolismo , Fosforilação , Plasmídeos/metabolismo , Presenilina-1 , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Transfecção , Ubiquitina/metabolismo
16.
J Steroid Biochem Mol Biol ; 94(1-3): 209-17, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15862968

RESUMO

We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.


Assuntos
Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Dactinomicina/farmacologia , Células HeLa , Temperatura Alta , Humanos , Cinética , Regiões Promotoras Genéticas , Transfecção
17.
EMBO J ; 23(13): 2586-96, 2004 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-15192701

RESUMO

Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by gamma-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.


Assuntos
Doença de Alzheimer/genética , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas tau/antagonistas & inibidores , Animais , Apoptose , Western Blotting , Caderinas/metabolismo , Carbamatos/farmacologia , Linhagem Celular Transformada , Sobrevivência Celular , Transformação Celular Viral , Dipeptídeos/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Proteínas de Membrana/genética , Camundongos/embriologia , Camundongos Knockout , Fosfatidilinositol 3-Quinases/química , Fosforilação , Testes de Precipitina , Presenilina-1 , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Simplexvirus/genética
18.
EMBO J ; 21(8): 1948-56, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11953314

RESUMO

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin- catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway.


Assuntos
Caderinas/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Junções Aderentes , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Sítios de Ligação , Caderinas/genética , Humanos , Líquido Intracelular , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Presenilina-1 , Processamento de Proteína Pós-Traducional
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