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1.
Mol Biotechnol ; 57(6): 513-25, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25663099

RESUMEN

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.


Asunto(s)
Pichia/genética , Ribonucleasas/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Cartilla de ADN , Glicosilación , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Ribonucleasas/química , Ribonucleasas/aislamiento & purificación , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/aislamiento & purificación
2.
Mol Pharmacol ; 83(3): 621-32, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23233488

RESUMEN

It has been proposed that OX(1) orexin receptors and CB(1) cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX(1) receptors and potentiate OX(1) receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX(1) receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX(1)-CB(1) receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX(1) receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB(1) receptor-expressing cells. When OX(1) and CB(1) receptors were expressed in the same cells, OX(1) stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX(1) response as such was fully abolished by specific inhibition of CB(1) receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX(1)-CB(1) heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX(1)-CB(1) synergism and thus suggest a functional rather than a molecular interaction of OX(1) and CB(1) receptors.


Asunto(s)
Endocannabinoides/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Comunicación Autocrina , Células CHO , Cricetinae , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glicéridos/metabolismo , Humanos , Lipoproteína Lipasa/antagonistas & inhibidores , Receptores de Orexina , Fosforilación/fisiología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Muscarínico M1/metabolismo , Transducción de Señal
3.
J Biol Chem ; 287(53): 44345-60, 2012 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-23144458

RESUMEN

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.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C-delta/metabolismo , Canales Catiónicos TRPC/metabolismo , Glándula Tiroides/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Regulación de la Expresión Génica , Homeostasis , Proteínas de la Membrana/genética , Proteína Quinasa C-delta/genética , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Molécula de Interacción Estromal 2 , Canales Catiónicos TRPC/genética , Glándula Tiroides/enzimología
4.
Mol Endocrinol ; 26(12): 2046-57, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23015753

RESUMEN

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.


Asunto(s)
Calcio/metabolismo , AMP Cíclico/metabolismo , Receptores de Tirotropina/metabolismo , Canales Catiónicos TRPC/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Canales Catiónicos TRPC/genética , Glándula Tiroides/citología
5.
Eur J Pharmacol ; 683(1-3): 63-70, 2012 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-22465183

RESUMEN

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.


Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 2/metabolismo , Venenos Elapídicos/metabolismo , Neurotoxinas/metabolismo , Péptidos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas de Reptiles/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Antagonistas de Receptores Adrenérgicos alfa 2/química , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Sitio Alostérico , Animales , Tartrato de Brimonidina , Señalización del Calcio/efectos de los fármacos , Línea Celular , Venenos Elapídicos/química , Humanos , Ligandos , Proteínas Mutantes Quiméricas/antagonistas & inhibidores , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/farmacología , Péptidos/química , Péptidos/genética , Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas , Quinolizinas/metabolismo , Quinolizinas/farmacología , Quinoxalinas/metabolismo , Quinoxalinas/farmacología , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas de Reptiles/química , Proteínas de Reptiles/genética , Proteínas de Reptiles/farmacología , Transducción de Señal/efectos de los fármacos , Spodoptera
6.
Biotechnol Appl Biochem ; 59(4): 314-21, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23586865

RESUMEN

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.


Asunto(s)
Baculoviridae/genética , Baculoviridae/fisiología , Ingeniería Genética/métodos , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Virus del Sarcoma de Rous/genética , Animales , Expresión Génica , Vectores Genéticos/genética , Humanos , Células Sf9 , Spodoptera
7.
Biochem Biophys Res Commun ; 417(1): 93-7, 2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22138651

RESUMEN

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.


Asunto(s)
Venenos Elapídicos/química , Glicosilfosfatidilinositoles/química , Antagonistas Muscarínicos/química , Neurotoxinas/química , Péptidos/química , Receptor Muscarínico M1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular , Venenos Elapídicos/genética , Venenos Elapídicos/farmacología , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Antagonistas Muscarínicos/farmacología , Neurotoxinas/genética , Neurotoxinas/farmacología , Péptidos/genética , Péptidos/farmacología , Ensayo de Unión Radioligante
8.
Toxins (Basel) ; 3(11): 1393-404, 2011 11.
Artículo en Inglés | MEDLINE | ID: mdl-22174976

RESUMEN

Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M(1) muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M(1) receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M(5) subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M(1) receptor. An M(5) receptor construct (M(5)-E(175)Y(184)E(474)), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent K(i) = 24 nM; apparent K(i) for M(1) = 0.5 nM). After screening for different domains and residues, we found a specific residue (P(179) to L in M(5)) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M(5)-EL(179)YE, apparent K(i) = 0.5 nM). Mutation of P(179) to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.


Asunto(s)
Venenos Elapídicos/metabolismo , Receptor Muscarínico M5/metabolismo , Animales , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M5/química , Receptor Muscarínico M5/genética , Células Sf9 , Spodoptera
9.
FEBS Lett ; 585(9): 1368-74, 2011 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-21510948

RESUMEN

We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuropéptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropéptido/agonistas , Proteínas Recombinantes de Fusión/agonistas , Secuencia de Aminoácidos , Sitios de Unión/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Neuropéptidos/química , Receptores de Orexina , Orexinas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Proteínas Recombinantes de Fusión/química
10.
Biotechnol Appl Biochem ; 58(1): 39-49, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21446958

RESUMEN

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.


Asunto(s)
Baculoviridae/metabolismo , Regulación Neoplásica de la Expresión Génica , Pichia/metabolismo , Ribonucleasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Baculoviridae/genética , Secuencia de Bases , Biocatálisis , Células Cultivadas , Clonación Molecular , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutación , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Proteínas Supresoras de Tumor/genética
11.
Cell Calcium ; 48(2-3): 114-23, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20728215

RESUMEN

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.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Proteínas de Homeodominio/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores de Neuropéptido/fisiología , Canales Catiónicos TRPC/fisiología , Calcio/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular Tumoral , Humanos , Neuroblastoma/química , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/metabolismo , Neuronas/patología , Receptores de Orexina , Proteína Quinasa C/fisiología , Receptores de Bradiquinina/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropéptido/genética , Rojo de Rutenio/farmacología , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/fisiología , Espermina/fisiología , Canales Catiónicos TRPC/antagonistas & inhibidores , Canal Catiónico TRPC6
12.
Endocr Relat Cancer ; 17(3): 785-96, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20576801

RESUMEN

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.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Superóxido Dismutasa/metabolismo , Neoplasias de la Tiroides/enzimología , Animales , Western Blotting , Calcio/metabolismo , Carcinoma , Carcinoma Papilar , Proliferación Celular , Regulación hacia Abajo , Humanos , Peróxido de Hidrógeno/metabolismo , Masculino , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Cáncer Papilar Tiroideo , Carcinoma Anaplásico de Tiroides , Glándula Tiroides/enzimología , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Células Tumorales Cultivadas
13.
Toxicon ; 56(3): 440-7, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20466015

RESUMEN

Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MTalpha with the human alpha(2B)-adrenoceptor. In a functional assay, measuring the alpha(2B)-adrenoceptor-induced increase in intracellular [Ca(2+)], we found that both venomous MTalpha and synthetic MTalpha inhibited the response in a concentration-dependent way. MTalpha did not affect the responses of alpha(2A)-, alpha(2C)-, alpha(1A)- or alpha(1B)-adrenoceptors. To further explore the binding of MTalpha to the alpha(2B)-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [(3)H]RX821002 as reporter ligand. MTalpha bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MTalpha displaced the radioligand with an IC(50) of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MTalpha suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to alpha(2B)-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MTalpha did not affect [(3)H]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block alpha(2B)-adrenoceptors, showing that the toxin is a more potent antagonist for the alpha(2B)-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of alpha(2)-adrenoceptor physiology.


Asunto(s)
Antagonistas Adrenérgicos alfa/farmacología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Animales , Línea Celular , Humanos , Ensayo de Unión Radioligante
14.
J Cell Physiol ; 221(1): 67-74, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19507192

RESUMEN

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.


Asunto(s)
Canales de Calcio/metabolismo , Diferenciación Celular , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Canales Catiónicos TRPM/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Isotiocianatos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Neuroblastoma/genética , Técnicas de Placa-Clamp , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPM/genética , Canales de Potencial de Receptor Transitorio/genética
15.
Neurosci Lett ; 457(1): 32-5, 2009 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-19429156

RESUMEN

Manganese in millimolar concentration caused increase in specific binding of [(3)H]8-OH-DPAT to rat hippocampal membranes up to 44% in comparison with experiments in the presence of Mg(2+), while no significant differences were found in rat cortical membranes. Similar increase in high-affinity agonist binding sites by Mn(2+) was found in displacement curves of 8-OH-DPAT, where antagonist [(3)H]WAY100635 was used as reporter ligand. The removal of bivalent ions with EDTA caused full loss of high-affinity binding of agonists, but not for antagonists. Therefore it was hypothesized, that the effect of Mn(2+)- and Mg(2+)-ions was modulated through their action on different G-proteins. Results showed that efficient coupling of G-protein and 5-HT(1A) receptors is crucial to modify Mg(2+) and Mn(2+) effects, whereas Mn(2+) is more potent stabilizer of agonist high-affinity binding, especially when GTPgammaS is present. Using Sf9 cells as model system, we have shown that G(i1) proteins are required to modulate Mn(2+)-dependent high-affinity agonist binding to 5-HT(1A) receptors, but further studies are necessary to find the cofactors of Mn(2+) modulation to signal transduction.


Asunto(s)
Membrana Celular/metabolismo , Hipocampo/metabolismo , Manganeso/administración & dosificación , Receptor de Serotonina 5-HT1A/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar
16.
J Neurosci ; 26(42): 10658-66, 2006 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-17050705

RESUMEN

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.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Diferenciación Celular/fisiología , Diglicéridos/farmacología , Neuroblastoma/metabolismo , Receptores de Neuropéptido/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Insectos , Magnesio/farmacología , Neuroblastoma/patología , Receptores de Orexina , Receptores Acoplados a Proteínas G
17.
Biochem Pharmacol ; 71(6): 827-36, 2006 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-16430869

RESUMEN

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.


Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Riñón/efectos de los fármacos , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Receptores de Neuropéptido/metabolismo , Baculoviridae/genética , Línea Celular , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/genética , Humanos , Riñón/metabolismo , Receptores de Orexina , Orexinas , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/agonistas , Transducción de Señal/efectos de los fármacos , Transducción Genética
18.
J Biol Chem ; 279(49): 50923-9, 2004 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-15452105

RESUMEN

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe --> Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent Ki = 0.26 nM in a functional assay) compared with the M1 receptor (apparent Ki = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.


Asunto(s)
Venenos Elapídicos/química , Venenos Elapídicos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/química , Línea Celular , Relación Dosis-Respuesta a Droga , Venenos Elapídicos/metabolismo , Ácido Glutámico/química , Humanos , Insectos , Cinética , Ligandos , Lisina/química , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Fenilalanina/química , Unión Proteica , Estructura Terciaria de Proteína , Receptor Muscarínico M1/química , Receptor Muscarínico M3/química , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Factores de Tiempo
19.
Neurochem Int ; 44(2): 119-24, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12971914

RESUMEN

The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPgammaS binding revealed no significant differences between M2/Gi and M2/Go complexes neither with respect to affinities nor efficacies of the ligands studied. Coexpression with either G protein caused constitutive activity of the receptor amounting up to 66% of stimulable [35S]GTPgammaS binding. Muscarinic antagonists, like atropine, scopolamine and N-methylscopolamine, behaved as inverse antagonists with potencies in good agreement with their binding affinities to the receptor. The results implicate that the functional reconstitution of M2 muscarinic receptor with either Gi or Go proteins in insect cells provides a valuable tool for screening of potencies as well as efficacies of agonists, partial agonists and inverse agonists at this receptor.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Agonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Animales , Baculoviridae/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Nucleótidos de Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Insectos , Ligandos , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante , Receptor Muscarínico M2
20.
J Neurochem ; 83(6): 1252-61, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12472880

RESUMEN

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.


Asunto(s)
Adenilil Ciclasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Humanos , Idazoxan/análogos & derivados , Idazoxan/farmacocinética , Toxina del Pertussis/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Receptores Adrenérgicos alfa 2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Spodoptera , Acetato de Tetradecanoilforbol/farmacología
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