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1.
Eur J Pharmacol ; 807: 1-11, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28438647

RESUMO

The α7 nicotinic acetylcholine receptor is thought to play an important role in human cognition. Here we describe the in vivo effects of BMS-902483, a selective potent α7 nicotinic acetylcholine receptor partial agonist, in relationship to α7 nicotinic acetylcholine receptor occupancy. BMS-902483 has low nanomolar affinity for rat and human α7 nicotinic acetylcholine receptors and elicits currents in cells expressing human or rat α7 nicotinic acetylcholine receptors that are about 60% of the maximal acetylcholine response. BMS-902483 improved 24h novel object recognition memory in mice with a minimal effective dose (MED) of 0.1mg/kg and reversed MK-801-induced deficits in a rat attentional set-shifting model of executive function with an MED of 3mg/kg. Enhancement of novel object recognition was blocked by the silent α7 nicotinic acetylcholine receptor agonist, NS6740, demonstrating that activity of BMS-902483 was mediated by α7 nicotinic acetylcholine receptors. BMS-902483 also reversed ketamine-induced deficits in auditory gating in rats, and enhanced ex vivo hippocampal long-term potentiation examined 24h after dosing in mice. Results from an ex vivo brain homogenate binding assay showed that α7 receptor occupancy ranged from 64% (novel object recognition) to ~90% (set shift and gating) at the MED for behavioral and sensory processing effects of BMS-902483.


Assuntos
Cognição/efeitos dos fármacos , Agonismo Parcial de Drogas , Agonistas Nicotínicos/farmacologia , Quinuclidinas/farmacologia , Filtro Sensorial/efeitos dos fármacos , Compostos de Espiro/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Atenção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Ratos
2.
Bioorg Med Chem ; 24(7): 1455-68, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26917221

RESUMO

N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.


Assuntos
Inibidores Enzimáticos/farmacologia , Glicina/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicina/análogos & derivados , Glicina/química , Humanos , Lipase Lipoproteica/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
3.
J Pharmacol Exp Ther ; 356(2): 293-304, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26582730

RESUMO

The muscarinic acetylcholine receptor subtype 1 (M1) receptors play an important role in cognition and memory, and are considered to be attractive targets for the development of novel medications to treat cognitive impairments seen in schizophrenia and Alzheimer's disease. Indeed, the M1 agonist xanomeline has been shown to produce beneficial cognitive effects in both Alzheimer's disease and schizophrenia patients. Unfortunately, the therapeutic utility of xanomeline was limited by cholinergic side effects (sweating, salivation, gastrointestinal distress), which are believed to result from nonselective activation of other muscarinic receptor subtypes such as M2 and M3. Therefore, drug discovery efforts targeting the M1 receptor have focused on the discovery of compounds with improved selectivity profiles. Recently, allosteric M1 receptor ligands have been described, which exhibit excellent selectivity for M1 over other muscarinic receptor subtypes. In the current study, the following three compounds with mixed agonist/positive allosteric modulator activities that are highly functionally selective for the M1 receptor were tested in rats, dogs, and cynomologous monkeys: (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1,4'-bipiperidine]-1'-carboxylate). Despite their selectivity for the M1 receptor, all three compounds elicited cholinergic side effects such as salivation, diarrhea, and emesis. These effects could not be explained by activity at other muscarinic receptor subtypes, or by activity at other receptors tested. Together, these results suggest that activation of M1 receptors alone is sufficient to produce unwanted cholinergic side effects such as those seen with xanomeline. This has important implications for the development of M1 receptor-targeted therapeutics since it suggests that dose-limiting cholinergic side effects still reside in M1 receptor selective activators.


Assuntos
Agonistas Muscarínicos/metabolismo , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Relação Dose-Resposta a Droga , Humanos , Macaca fascicularis , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley
4.
J Pharmacol Exp Ther ; 354(3): 340-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109678

RESUMO

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Animais , Células CHO , Linhagem Celular , Células Cultivadas , Cricetulus , Células HEK293 , Humanos , Camundongos , Ratos , Esquizofrenia/tratamento farmacológico
6.
Bioorg Med Chem Lett ; 24(6): 1611-4, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24507922
7.
Bioorg Med Chem ; 22(5): 1782-90, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24495863

RESUMO

Comprehensive structure activity relationship (SAR) studies were conducted on a focused screening hit, 2-(methylthio)-3-(phenylsulfonyl)-4H-pyrido[1,2-a]pyrimidin-4-imine (1, IC50: 4.0 nM), as 5-HT6 selective antagonists. Activity was improved some 2-4 fold when small, electron-donating groups were added to the central core as observed in 19, 20 and 26. Molecular docking of key compounds in a homology model of the human 5-HT6 receptor was used to rationalize our structure-activity relationship (SAR) findings. In pharmacokinetic experiments, compound 1 displayed good brain uptake in rats following intra-peritoneal administration, but limited oral bioavailability.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Iminas/farmacocinética , Receptores de Serotonina/uso terapêutico , Animais , Humanos , Iminas/farmacologia , Simulação de Acoplamento Molecular , Ratos , Relação Estrutura-Atividade
8.
J Biomol Screen ; 19(4): 595-605, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24241710

RESUMO

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Lipase Lipoproteica/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato
9.
J Neurochem ; 129(2): 275-83, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24266811

RESUMO

NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co-agonists, glycine or D-serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D-serine have shown positive results, although concerns over toxicity related to the high-doses required for efficacy remain. Synaptic concentrations of D-serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter-1 (asc-1). Inhibition of asc-1 would increase synaptic D-serine and possibly glycine, eliminating the need for high-dose systemic D-serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS-466442), the first known small molecule inhibitor of asc-1. Compound 1 selectively inhibited asc-1 mediated D-serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc-1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc-1 transporter and may provide a new path for the development of asc-1 inhibitors for the treatment of schizophrenia.


Assuntos
Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Agonistas de Aminoácidos Excitatórios/farmacologia , Histidina/análogos & derivados , Indóis/síntese química , Indóis/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Aminoácidos/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Glicina/metabolismo , Histidina/síntese química , Histidina/farmacologia , Humanos , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Bibliotecas de Moléculas Pequenas , Sinaptossomos/metabolismo
10.
J Med Chem ; 54(19): 6548-62, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21882820

RESUMO

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an "open" conformation or a "closed" conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced "superopen" conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.


Assuntos
Modelos Moleculares , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/antagonistas & inibidores , Tiofenos/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Relação Estrutura-Atividade , Tiofenos/síntese química
11.
J Physiol ; 589(Pt 20): 4857-84, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21807615

RESUMO

Depolarization-induced suppression of inhibition (DSI) is a prevailing form of endocannabinoid signalling. However, several discrepancies have arisen regarding the roles played by the two major brain endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide, in mediating DSI. Here we studied endocannabinoid signalling in the prefrontal cortex (PFC), where several components of the endocannabinoid system have been identified, but endocannabinoid signalling remains largely unexplored. In voltage clamp recordings from mouse PFC pyramidal neurons, depolarizing steps significantly suppressed IPSCs induced by application of the cholinergic agonist carbachol. DSI in PFC neurons was abolished by extra- or intracellular application of tetrahydrolipstatin (THL), an inhibitor of the 2-AG synthesis enzyme diacylglycerol lipase (DAGL). Moreover, DSI was enhanced by inhibiting 2-AG degradation, but was unaffected by inhibiting anandamide degradation. THL, however, may affect other enzymes of lipid metabolism and does not selectively target the α (DAGLα) or ß (DAGLß) isoforms of DAGL. Therefore, we studied DSI in the PFC of DAGLα(-/-) and DAGLß(-/-) mice generated via insertional mutagenesis by gene-trapping with retroviral vectors. Gene trapping strongly reduced DAGLα or DAGLß mRNA levels in a locus-specific manner. In DAGLα(-/-) mice cortical levels of 2-AG were significantly decreased and DSI was completely abolished, whereas DAGLß deficiency did not alter cortical 2-AG levels or DSI. Importantly, cortical levels of anandamide were not significantly affected in DAGLα(-/-) or DAGLß(-/-) mice. The chronic decrease of 2-AG levels in DAGLα(-/-) mice did not globally alter inhibitory transmission or the response of cannabinoid-sensitive synapses to cannabinoid receptor stimulation, although it altered some intrinsic membrane properties. Finally, we found that repetitive action potential firing of PFC pyramidal neurons suppressed synaptic inhibition in a DAGLα-dependent manner. These results show that DSI is a prominent form of endocannabinoid signalling in PFC circuits. Moreover, the close agreement between our pharmacological and genetic studies indicates that 2-AG synthesized by postsynaptic DAGLα mediates DSI in PFC neurons.


Assuntos
Ácidos Araquidônicos/fisiologia , Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Glicerídeos/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Lipase Lipoproteica/fisiologia , Inibição Neural/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Isoenzimas/deficiência , Isoenzimas/fisiologia , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Técnicas de Patch-Clamp , Alcamidas Poli-Insaturadas , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-21795790

RESUMO

Protein tyrosine phosphatase γ is a membrane-bound receptor and is designated RPTPγ. RPTPγ and two mutants, RPTPγ(V948I, S970T) and RPTPγ(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.


Assuntos
Domínio Catalítico , Proteínas Tirosina Fosfatases Semelhantes a Receptores/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/isolamento & purificação
13.
Biochem Biophys Res Commun ; 411(4): 809-14, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21787747

RESUMO

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.


Assuntos
Lipase Lipoproteica/química , Catálise , Membrana Celular/enzimologia , Compostos Cromogênicos/química , Cicloexanonas/química , Fluorescência , Células HEK293 , Humanos , Lactonas/química , Lipase Lipoproteica/genética , Mutação , Orlistate , Especificidade por Substrato
14.
BMC Neurosci ; 11: 143, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-21054826

RESUMO

BACKGROUND: Accumulation of amyloid-ß (Aß) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aß, but not all are equally tractable for drug discovery. RESULTS: To search for novel targets that affect brain Aß without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aß ELISA assays. Although robust Aß lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aß, including a GPR3 KO strain, which had previously been proposed as an Aß target. However, significantly increased Aß was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT). CONCLUSIONS: Thus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aß levels in the brain are rare.


Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/genética , Técnicas de Inativação de Genes/métodos , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Testes Genéticos/métodos , Manosiltransferases/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pentosiltransferases/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo
15.
Mol Pharmacol ; 69(4): 1396-404, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16399850

RESUMO

Neurotrophins are a family of secreted proteins that play an important role in the development, differentiation, and survival of neurons. Studies also suggest that aberrant neurotrophin signaling may play a role in processes underlying disease states such as schizophrenia, Alzheimer's disease, and depression. Whereas the development of agents that selectively stimulate neurotrophin signaling has proven to be difficult, compounds have been identified that potentiate neurotrophin 3 (NT-3)-mediated activation of trk A. In the present studies, we extend those initial observations to identify compounds that also potentiate NT-3-mediated activation of trk B. Compound potentiation of NT-3 was observed using several readouts of transfected and endogenous trk receptor activity, including trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation, reporter assay activity (beta-lactamase and luciferase), cell survival and neurite extension assays. Studies using chimeric trk receptors demonstrated that the extracellular domain is essential for compound potentiation and rule out interaction with intracellular signaling molecules as a mechanism of compound activity. Thus, the present studies demonstrate that trk B receptor activity can be potentiated by small-molecule compounds via the extracellular domain of the receptor and provide reagents for further evaluating the role of NT-3-mediated trk A and trk B activity in vivo.


Assuntos
Neurotrofina 3/farmacologia , Receptor trkB/agonistas , Animais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , DNA Complementar , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais
17.
Protein Expr Purif ; 31(1): 19-33, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12963337

RESUMO

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.


Assuntos
Domínio Catalítico , Fosfoproteínas Fosfatases/isolamento & purificação , Fosfoproteínas/metabolismo , Animais , Resinas de Troca de Ânions/química , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Western Blotting , Encéfalo/enzimologia , Química Encefálica , Células COS , Caseínas/metabolismo , Bovinos , Cromatografia de Afinidade , Clonagem Molecular , Éteres Cíclicos/farmacologia , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/química , Fígado/enzimologia , Masculino , Camundongos , Microcistinas , Dados de Sequência Molecular , Miocárdio/química , Miocárdio/enzimologia , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilase a/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica/fisiologia , Proteínas Quinases/metabolismo , Proteína Fosfatase 2 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Resinas Sintéticas , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
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