RESUMO
Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.
Assuntos
Pichia/genética , Ribonucleases/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Primers do DNA , Glicosilação , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ribonucleases/química , Ribonucleases/isolamento & purificação , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/isolamento & purificaçãoRESUMO
Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCß1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca(2+)-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca(2+)-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C-delta/metabolismo , Canais de Cátion TRPC/metabolismo , Glândula Tireoide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Homeostase , Proteínas de Membrana/genética , Proteína Quinase C-delta/genética , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Molécula 2 de Interação Estromal , Canais de Cátion TRPC/genética , Glândula Tireoide/enzimologiaRESUMO
Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Receptores da Tireotropina/metabolismo , Canais de Cátion TRPC/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/metabolismo , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno , Ratos , Canais de Cátion TRPC/genética , Glândula Tireoide/citologiaRESUMO
Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α(2B)-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [³H]UK14,304 agonist and the [³H]MK-912 antagonist binding to the α(2B)-adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of B(max) without change in radioligand affinity. On the contrary, cellular signaling via the α(2B)-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α(2B)- and α(2A)- or α(2C)-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α(2B) for α(2A)-adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α(2B)-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α(2B)-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/metabolismo , Venenos Elapídicos/metabolismo , Neurotoxinas/metabolismo , Peptídeos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas de Répteis/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/química , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Sítio Alostérico , Animais , Tartarato de Brimonidina , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Venenos Elapídicos/química , Humanos , Ligantes , Proteínas Mutantes Quiméricas/antagonistas & inibidores , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/farmacologia , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Quinolizinas/metabolismo , Quinolizinas/farmacologia , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas de Répteis/química , Proteínas de Répteis/genética , Proteínas de Répteis/farmacologia , Transdução de Sinais/efeitos dos fármacos , SpodopteraRESUMO
Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7-GPI remained anchored to the cell surface and selectively inhibited M(1) muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.
Assuntos
Venenos Elapídicos/química , Glicosilfosfatidilinositóis/química , Antagonistas Muscarínicos/química , Neurotoxinas/química , Peptídeos/química , Receptor Muscarínico M1/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Venenos Elapídicos/genética , Venenos Elapídicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Antagonistas Muscarínicos/farmacologia , Neurotoxinas/genética , Neurotoxinas/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Ensaio RadioliganteRESUMO
The effect of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter on expression levels of complementary DNAs (cDNAs) encoding seven transmembrane receptors was studied using the baculovirus expression vector system. Expression of the human α(2B)-adrenoceptor (AR) cDNA under the control of the polyhedrin (POL) promoter produced up to 7.6 pmol/mg protein at 28 H post infection (p.i.) in Sf9 cells. The addition of the RSV promoter increased the expression to 11.6 pmol/mg protein. Dramatic increases in expression levels at early times were also obtained with the α(2A)-AR, the M1 and M4 muscarinic receptors, and the orexin OX1 receptor. Analysis of the time-dependent expression revealed that expression driven by the RSV promoter reaches almost maximum 24 H p.i. and that this promoter is superior to the often used POL promoter at early times p.i. when functional studies need to be performed. Functional enhancement of signaling as a result of early expression is demonstrated with the α(2B)-AR and the OX1 receptor. Finally, enhanced green fluorescent protein fluorescence in living cells was used to monitor expression by various viral promoters. The results verified the early transcriptional activity of the RSV promoter, whereas the cytomegalovirus promoter was found to be poorly active in Sf9 cells.
Assuntos
Baculoviridae/genética , Baculoviridae/fisiologia , Engenharia Genética/métodos , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/genética , Vírus do Sarcoma de Rous/genética , Animais , Expressão Gênica , Vetores Genéticos/genética , Humanos , Células Sf9 , SpodopteraRESUMO
We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.
Assuntos
Baculoviridae/metabolismo , Regulação Neoplásica da Expressão Gênica , Pichia/metabolismo , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Baculoviridae/genética , Sequência de Bases , Biocatálise , Células Cultivadas , Clonagem Molecular , Glicosilação , Humanos , Dados de Sequência Molecular , Mutação , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
TRPC channels play significant roles in the regulation of neuronal plasticity and development. The mechanism by which these nonselective cation channels exert their trophic actions appears to involve entry of Ca(2+) into the cells. Using a neuronal cell model (differentiated human IMR32 neuroblastoma cells), we demonstrate a central role for sodium entry via TRPC3/6 channels in receptor-mediated increases in intracellular calcium. These Na(+)-dependent Ca(2+) influxes, which were observed in a subpopulation of cells, were efficiently blocked by protein kinase C activation, by the Na(+)/Ca(2+) exchanger inhibitors, and by molecular disruption of TRPC3/6 channel function. On the other hand, another subpopulation of cells showed a Na(+)-independent Ca(2+) entry upon stimulation of the same receptors, orexin/hypocretin and bradykinin receptors. This second type of response was not affected by the above mentioned treatments, but it was sensitive to polyvalent cations, such as ruthenium red, spermine and Gd(3+). The data suggest that a NCX-TRPC channel interaction constitutes an important functional unit in receptor-mediated Ca(2+) influx in neuronal cells.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Proteínas de Homeodomínio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Neuropeptídeos/fisiologia , Canais de Cátion TRPC/fisiologia , Cálcio/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Neuroblastoma/química , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptores de Orexina , Proteína Quinase C/fisiologia , Receptores da Bradicinina/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Rutênio Vermelho/farmacologia , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/fisiologia , Espermina/fisiologia , Canais de Cátion TRPC/antagonistas & inibidores , Canal de Cátion TRPC6RESUMO
Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.
Assuntos
Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Superóxido Dismutase/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Animais , Western Blotting , Cálcio/metabolismo , Carcinoma , Carcinoma Papilar , Proliferação de Células , Regulação para Baixo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxidos/metabolismo , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide , Glândula Tireoide/enzimologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.
Assuntos
Canais de Cálcio/metabolismo , Diferenciação Celular , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Canais de Cátion TRPM/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Isotiocianatos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Técnicas de Patch-Clamp , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPM/genética , Canais de Potencial de Receptor Transitório/genéticaRESUMO
We studied the cellular response to orexin type 1 receptor (OX1R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2'-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX1R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca2+ concentration, [Ca2+]i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca2+, by application of a high concentration (10 mM) of Mg2+, and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX1R response with regard to Mg2+ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX1R stimulation. The results suggest that the OX1R activates a Ca2+ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Diglicerídeos/farmacologia , Neuroblastoma/metabolismo , Receptores de Neuropeptídeos/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Insetos , Magnésio/farmacologia , Neuroblastoma/patologia , Receptores de Orexina , Receptores Acoplados a Proteínas GRESUMO
The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Rim/efeitos dos fármacos , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Receptores de Neuropeptídeos/metabolismo , Baculoviridae/genética , Linhagem Celular , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/genética , Humanos , Rim/metabolismo , Receptores de Orexina , Orexinas , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução GenéticaRESUMO
The effect of Gi/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed alpha2A-adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin-sensitive manner. Activation of Gs proteins switched the response of the alpha2A-adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbetagamma scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (alphao1 + beta1gamma2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (alphai1 + beta1gamma2) resulted in a potentiation of both the TPA- and the Gs-stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein-linked signalling.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Humanos , Idazoxano/análogos & derivados , Idazoxano/farmacocinética , Toxina Pertussis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Spodoptera , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The Sf9 insect cell line, derived from Spodoptera frugiperda, was used to study the regulation of adenylate cyclase (AC) activity by octopamine receptors. The cyclic AMP (cAMP) production was stimulated in a concentration-dependent manner by octopamine. Octopamine also elicited a rise in cytosolic Ca(2+). The Ca(2+) elevation was independent of the cAMP elevation whereas the cAMP elevation was partially inhibited by removal of Ca(2+). The antagonistic effects of a series of compounds were tested on both responses. Phentolamine inhibited both responses with similar potency. Two of the tested compounds, MK-912 and RS 79948, were over 1000-fold more potent in blocking the Ca(2+) response. Ionomycin, a Ca(2+) ionophore, or activation of the heterologously expressed muscarinic M(3) receptors in the cells did not alone stimulate cAMP production. However, a Ca(2+) elevation potentiated cAMP production in the presence of a primary stimulant such as forskolin or activated G(s) proteins. This type of regulation of AC is different from previously identified Ca(2+)-sensitive AC isoforms. For comparison the Ca(2+)/calmodulin-activated type I AC was expressed and demonstrated to be stimulated directly by an increase in Ca(2+). Together the results demonstrate that octopamine can synergistically regulate the AC activity via two different receptors in Sf9 cells.