RESUMEN
We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein-coupled receptor, the bradykinin B2 receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein fluorescence measuring the accelerated rate of pH(i) recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B2 receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kinase C and phosphatidylinositol 3'-kinase. Immunoprecipitation studies showed that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. CaM appears to be a direct substrate for phosphorylation by Jak2 as measured by an in vitro kinase assay. We propose that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of CaM and most likely by increasing the binding of CaM to NHE-1.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Bradiquinina/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Bradiquinina/farmacología , Línea Celular , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Janus Quinasa 2 , Médula Renal/citología , Médula Renal/fisiología , Cinética , Ratones , Ratones Transgénicos , Modelos Biológicos , Toxina del Pertussis , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/genética , Transducción de Señal/fisiología , Virus 40 de los Simios/genética , Sodio/metabolismo , Transcripción Genética , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.
Asunto(s)
Receptores de Serotonina/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/farmacología , AMP Cíclico/metabolismo , Humanos , Canales Iónicos/fisiología , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/farmacología , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/farmacologíaRESUMEN
We constructed an expression vector for a fusion protein [ANG II type 1a receptor-green fluorescent protein (AT(1a)R-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat AT(1a)R. Chinese hamster ovary (CHO) cells transfected with AT(1a)R-GFP demonstrated specific, high-affinity (125)I-labeled ANG II binding (IC(50) 21 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with AT(1a)R or AT(1a)R-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT(1)R blocker losartan. ANG II-driven internalization of AT(1a)R-GFP in transfected CHO cells was demonstrated both by radioligand binding and by laser scanning confocal microscopy. Colocalization of GFP fluorescence with that of the nuclear stain TOTO-3 in confocal images was increased more than twofold after 1 h of ANG II exposure. We conclude that AT(1a)R-GFP exhibits similar pharmacological behavior to that of the native AT(1a)R. Our observations also support previous evidence for the presence of AT(1a)R in the nucleus and suggest that the density of AT(1a)R in the nucleus may be regulated by exposure to its ligand.
Asunto(s)
Núcleo Celular/metabolismo , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Células CHO , Calcio/metabolismo , Cricetinae , Colorantes Fluorescentes , Genes Reporteros , Proteínas Fluorescentes Verdes , Radioisótopos de Yodo , Losartán/farmacología , Microscopía Confocal , Compuestos de Quinolinio , Ensayo de Unión Radioligante , Ratas , Receptores de Angiotensina/agonistas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TiazolesRESUMEN
Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.
Asunto(s)
Mesangio Glomerular/metabolismo , Peróxido de Hidrógeno/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Serotonina/fisiología , Animales , Células Cultivadas , Activación Enzimática , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Mesangio Glomerular/citología , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Fosforilación , Proteína Quinasa C/fisiología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/fisiología , Receptor de Serotonina 5-HT2A , Serotonina/farmacología , Fosfolipasas de Tipo C/fisiología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/fisiología , Receptores de Serotonina/fisiología , Acetilcisteína/farmacología , Animales , Células CHO , Cricetinae , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Cinética , NADPH Oxidasas , Oxidación-Reducción , Fosforilación , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/metabolismo , Serotonina/farmacología , Superóxidos/metabolismo , Transfección , Factores de Virulencia de Bordetella/farmacología , Dominios Homologos srcRESUMEN
The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identification of its cDNA.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Receptores de Serotonina/fisiología , Transducción de Señal/fisiología , Transfección/fisiología , Alelos , Animales , Células HeLa , Humanos , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.
Asunto(s)
Músculo Liso Vascular/metabolismo , Receptores de Tromboxanos/metabolismo , Animales , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células CHO , Células COS , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario/biosíntesis , Ácidos Grasos Insaturados/farmacología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Hidrazinas/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/genética , TransfecciónRESUMEN
We examined the links between fibrotic and proliferative pathways for the 5-HT2A receptor in rat mesangial cells. Serotonin (5-hydroxytryptamine, 5-HT) induced transforming growth factor-beta1 (TGF-beta1) mRNA in a concentration-dependent (peak at 30 nM 5-HT) and time-dependent fashion. For 10 nM 5-HT, the effect was noticeable at 1 h and maximal by 6 h. Inhibition of 1) protein kinase C (PKC), 2) mitogen- and extracellular signal-regulated kinase kinase (MEK1) with 2'-amino-3'-methoxyflavone (PD-90859), and 3) extracellular signal-regulated kinase (ERK) with apigenin attenuated this effect. The effect was blocked by antioxidants, N-acetyl-L-cysteine (NAC) and alpha-lipoic acid, and mimicked by direct application of H2O2. TGF-beta1 mRNA induction was also blocked by diphenyleneiodonium and 4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibit NAD(P)H oxidase, a source of oxidants. 5-HT increased the amount of TGF-beta1 protein, validating the mRNA studies and demonstrating that 5-HT potently activates ERK and induces TGF-beta1 mRNA and protein in mesangial cells. Mapping studies strongly supported relative positions of the components of the signaling cascade as follow: 5-HT2A receptor --> PKC --> NAD(P)H oxidase/reactive oxygen species --> MEK --> ERK --> TGF-beta1 mRNA. These studies demonstrate that mitogenic signaling components (PKC, MEK, and oxidants) are directly linked to the regulation of TGF-beta1, a key mediator of fibrosis. Thus a single stimulus can direct both proliferative and fibrotic signals in renal mesangial cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Mesangio Glomerular/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Receptores de Serotonina/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , División Celular/fisiología , Fibrosis , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Mesangio Glomerular/citología , MAP Quinasa Quinasa 1 , Masculino , NADPH Oxidasas/metabolismo , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genética , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.
Asunto(s)
Adenoviridae , Separación Celular/métodos , Técnicas de Transferencia de Gen , Mesangio Glomerular/citología , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Animales , Factor Natriurético Atrial/metabolismo , Factor Natriurético Atrial/farmacología , ADN Complementario , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes , Radioisótopos de Yodo , Liposomas/genética , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Transfección , beta-Galactosidasa/genéticaRESUMEN
Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Endocitosis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Serotonina/metabolismo , Animales , Células CHO , Calcio/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Calmodulina/antagonistas & inhibidores , Línea Celular , Clatrina/metabolismo , Cricetinae , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/metabolismoRESUMEN
It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.
Asunto(s)
Amilorida/análogos & derivados , Colecistoquinina/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Sodio/metabolismo , Amilorida/farmacología , Animales , Antígenos Transformadores de Poliomavirus/genética , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Canales de Calcio Tipo L , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/fisiología , Diltiazem/farmacología , Concentración de Iones de Hidrógeno , Insulina/genética , Neoplasias Intestinales , Ratones , Ratones Transgénicos , Poliomavirus/genética , Potasio/metabolismo , Canales de Potasio/fisiología , Regiones Promotoras Genéticas , Ratas , Virus 40 de los Simios/genéticaRESUMEN
These experiments tested the hypothesis that signalling elements involved in the activation of the extracellular signal-regulated protein kinase (ERK) mediate rapid activation of sodium-proton exchange (NHE) in fibroblasts when both signals are initiated by a single G protein-coupled receptor, the 5-HT1A receptor. Similarities between the two processes were comparable concentration-response curves and time-courses, and overlapping sensitivity to some pharmacological inhibitors of tyrosine kinases (staurosporine and genistein), and phosphoinositide 3'-kinase (wortmannin and LY204002). Activation of NHE was much more sensitive to the phosphatidylcholine-specific phospholipase inhibitor (D609) than was ERK. Neither pathway was sensitive to manoeuvres designed to block PKC. In contrast, Src or related kinases appear to be required to activate ERK, but not NHE. Transfection of cDNA constructs encoding inactive mutant phosphoinositide 3'-kinase, Grb2, Sos, Ras, and Raf molecules were successful in attenuating ERK, but had essentially no effect upon NHE activation. Finally, PD98059, an inhibitor of mitogen activated/extracellular signal regulated kinase kinase, blocked ERK but not NHE activation. Thus, in CHO fibroblast cells, activation by the 5-HT1A receptor of ERK and NHE share a number of overlapping features. However, our studies do not support a major role for ERK, when activated by the 5-HT1A receptor, as a short-term upstream regulator of NHE activity.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores de Serotonina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células CHO , Cricetinae , Fibroblastos , Humanos , Fosfolípidos/fisiología , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Serotonina 5-HT1 , Transducción de SeñalRESUMEN
5-HT1A receptors couple to many signaling pathways in CHO-K1 cells through pertussis toxin-sensitive G proteins. The purpose of this study was to determine which members of the Gi/o/z family mediate 5-HT1A receptor-activated Na+/H+ exchange as measured by microphysiometry of cell monolayers. The method was extensively validated, showing that proton efflux was sodium-dependent, inhibited by amiloride analogs, and activated by growth factors, phorbol ester, calcium ionophore, and hypertonic stress. 5-HT and the specific agonist (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide rapidly stimulated proton efflux that was blocked by a specific receptor antagonist, amiloride analogs or pertussis toxin. The activation by 5-HT depended upon extracellular sodium and could be demonstrated under conditions of imposed intracellular acid load, as well as in the presence and absence of glycolytic substrate. Acceleration of proton efflux was not inhibited by sequestration of G protein betagamma-subunits, a maneuver that blocked 5-HT1A receptor activation of mitogen-activated protein kinase. Transfection of Gzalpha and pertussis toxin-resistant mutants of Goalpha and Gialpha1 did not reverse the blockade induced by pertussis toxin. In contrast, pertussis toxin-resistant mutants of Gialpha2 and Gialpha3 "rescued" the ability of 5-HT to increase proton efflux after pertussis toxin treatment. These experiments demonstrate clearly that Gialpha2 and Gialpha3 can specifically mediate rapid agonist-induced acceleration of Na+/H+ exchange.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Serotonina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Células CHO , Cricetinae , Toxina del Pertussis , Unión Proteica , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Serotonin (5-HT) is a potent mitogen in many cells types, an action which is frequently mediated through pertussis toxin-sensitive G proteins. In the current study, we used pharmacological inhibitors and dominant negative signaling constructs to delineate elements which participate in the activation of MAPK, a growth-associated mitogen-activated protein kinase, by human G protein-coupled 5-HT1A receptor transfected into CHO-K1 cells in a stable manner. The activation pathway does not directly involve phorbol ester-sensitive protein kinase C types, but does require (i) pertussis toxin-sensitive G protein beta gamma-subunits, (ii) a staurosporine- and genistein-sensitive protein kinase, (iii) phosphoinositide-3'-kinase activity, (iv) activation of Sos in a multimolecular complex that contains p46Shc, and p52Shc, and Grb2, (v) the GTPase p21Ras, and (vi) the protein kinase p74Raf-1. These data demonstrate that the 5-HT1A receptor mediates MAPK activity by convergence upon a common activation pathway that is shared with receptor tyrosine kinases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Serotonina/metabolismo , Animales , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , Cricetinae , Activación Enzimática/efectos de los fármacos , Proteína Adaptadora GRB2 , Regulación de la Expresión Génica/genética , Immunoblotting , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Toxina del Pertussis , Pruebas de Precipitina , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transfección/genética , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The human 5-HT1A receptor was expressed in Sf9 insect cells to examine desensitization as manifested by agonist-induced uncoupling from G proteins and second messengers. New binding sites were detected after infection of cells with the 5-HT1A receptor-bearing baculovirus. 5-HT1A receptor agonists caused inhibition of cAMP accumulation that could be attenuated by specific receptor antagonists. Brief pretreatment with 5-HT resulted in (1) an uncoupling of receptor from G proteins as evidenced by a loss of high-affinity agonist binding sites and a diminished ability of the receptor to increase incorporation of AA-GTP into endogenous Go alpha-like G proteins, (2) a decreased ability of the receptor to inhibit cAMP accumulation, and (3) increased phosphorylation of the 5-HT1A receptor on serine and threonine residues. Phosphorylation occurred in the presence of a number of cyclic nucleotide dependent kinase inhibitors, and desensitization of the cAMP response occurred in the presence of H-7 and also in cells with prolonged exposure to PMA. Both phosphorylation and desensitization were markedly attenuated by 100 nM and 1 microM heparin and demonstrated similar time courses and concentration-response relationships. Those results demonstrate a close association between agonist-induced desensitization and phosphorylation of the 5-HT1A receptor in Sf9 cells through a pathway that mainly does not involve protein kinase A or C and might involve a G protein-linked receptor kinase.
Asunto(s)
Receptores de Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Baculoviridae/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , AMP Cíclico/biosíntesis , Cartilla de ADN/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Serotonina/farmacología , SpodopteraRESUMEN
Second messenger coupling of the 5-hydroxytryptamine (5-HT)2A receptor endogenous to cultured rat glomerular mesangial cells was studied. 5-HT induced an increase in total inositol phosphate levels (EC50 = 265 +/- 55 nM, maximum stimulation = 150 +/- 23%). That effect was sensitive to antagonists of the 5-HT2A receptor and was insensitive to pertussis toxin at doses that eliminated detectable pertussis toxin substrate, as determined by membrane ADP-ribosylation. Surprisingly, 5-HT also induced an inhibition of forskolin-stimulated cAMP accumulation (55 +/- 6%, IC50 = 5 +/- 3 nM). This effect was competitively antagonized by the 5-HT2A receptor antagonists ketanserin, ritanserin, and spiperone and could be produced by the 5-HT2 receptor agonists alpha-methyl-5-HT (66 +/- 13%, IC50 = 23 +/- 14 nM) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (65 +/- 4%, IC50 = 14 +/- 7 nM). The inhibition of cAMP accumulation occurred in the presence of a number of agents that either stimulate or inhibit protein kinase C activity, arachidonic acid metabolism, or Ca2+ mobilization. In isolated membranes, 5-HT induced a 36 +/- 5% inhibition of adenylyl cyclase activity (IC50 = 8 +/- 4 nM). Inhibition of cAMP accumulation in intact cells and of adenylyl cyclase activity in washed membranes was (> 50%) sensitive to pertussis toxin, implicating Gi alpha or Go alpha subunits in the inhibitory signal. These data suggest that the 5-HT2A receptor can be permissive in its coupling to G proteins and second messengers.
Asunto(s)
AMP Cíclico/antagonistas & inhibidores , Mesangio Glomerular/metabolismo , Receptores de Serotonina/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Mesangio Glomerular/citología , Mesangio Glomerular/enzimología , Proteína Quinasa C/metabolismo , Ratas , Serotonina/farmacología , Transducción de SeñalRESUMEN
Previous studies have demonstrated the presence of mitogenic serotonin [i.e., 5-hydroxytryptamine (5-HT)] receptors on glomerular mesangial cells and have linked those receptors to a complicated array of intracellular and autocrine/paracrine signaling pathways [T. Knauss and H. E. Abboud. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F844-F850, 1986; and N. Takuwa, M. Ganz, Y. Takuwa, R. B. Sterzel, and H. Rasmussen. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F431-F439, 1989]. Those studies suggested that the mesangial subtype of 5-HT receptor is a member of the 5-HT2 receptor family, which consists of three known members, designated as subtypes A, B, and C. The purpose of the current study was to identify the subtype of 5-HT2 receptor present on mesangial cells. Northern blot showed detectable mRNA for a putative 5-HT2A receptor, but not for 5-HT1A or 5-HT2C receptors. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides derived from the putative third and sixth transmembrane domains of cloned 5-HT2 receptors yielded a 580-nucleotide (nt) fragment. RT-PCR with primers highly specific for the 5-HT2A receptor and designed to amplify > 95% of its coding block yielded a product of 1,320 nt. Nested PCR reactions yielded products of the predicted sizes for the 5-HT2A receptor. Partial sequence information was obtained, and the sequence corresponded exactly (627/627 nt) to that published for the cloned rat brain 5-HT2A receptor. These studies identify the mesangial cell mitogenic 5-HT receptor as a 5-HT2A receptor subtype.
Asunto(s)
Mesangio Glomerular/metabolismo , Receptores de Serotonina/biosíntesis , Animales , Secuencia de Bases , Northern Blotting , Membrana Celular/metabolismo , Células Cultivadas , ADN/química , ADN/metabolismo , Cartilla de ADN , ADN Complementario , Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/química , Moldes GenéticosRESUMEN
Insect cell expression systems are used to characterize signaling components such as G protein-coupled receptors. As such, one must know whether endogenous G proteins couple to non-native receptors. We examined G protein linkages after infection of Sporodoptera frugiperda (Sf9) cells with a baculovirus encoding the 5-HT1A receptor. Receptor expression was confirmed by immunoblot. Some of the receptors were functional, showing guanine nucleotide-sensitive binding to the specific agonist ligand [3H]8-hydroxy-2-(di-n-propylamino)-1,2,3,4-tetranaphthalene). Peak expression (approximately 150 fmol/mg of membrane protein) was attained approximately 72-96 h post-infection. 5-HT-increased covalent binding of [32P]GTP-azidoanilide to a 40 kDa band, which was identified as a G protein by nucleotide blocking, Mg2+ dependence, and immunoblot and immunoprecipitation studies. The band comigrated with 1) pertussis toxin substrate(s), and 2) a band recognized by two G(o) alpha antisera and one common to heterotrimeric G protein alpha-subunits, but not by sera specific for Gs alpha or G(i) alpha. Labeled species could be precipitated with a G(o) alpha antiserum. 5-HT-increased labeling of the band was prevented by preincubation with pertussis toxin. These studies suggest that the 5-HT1A receptor couples effectively to native insect cell G(o)-like proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Serotonina/metabolismo , Secuencia de Aminoácidos , Anilidas , Animales , Baculoviridae/genética , Células Cultivadas , Clonación Molecular , Nucleótidos de Guanina/metabolismo , Humanos , Datos de Secuencia Molecular , Mariposas Nocturnas , Receptores de Serotonina/genéticaRESUMEN
The insertion of visual opsin into membranes occurred during in vitro translation of opsin mRNA in wheat germ extract in the presence of either microsomes or liposomes. The rhodopsin that integrated into both types of membranes after regeneration with 11-cis-retinal was functionally active (in contrast to the nonincorporated protein). Opsin either cotranslationally translocated into microsomes or inserted into liposomes had equal sensitivity to proteolysis and yielded the same pattern of peptides, which differed substantially from the set of peptides produced during proteolysis of opsin not incorporated into membranes. Thus visual opsin does not require protein translocation machinery for proper insertion into the lipid bilayer.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Corteza Cerebral/metabolismo , Membrana Dobles de Lípidos , Microsomas/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Rodopsina/genética , Animales , Bovinos , Proteínas del Ojo/genética , Fragmentos de Péptidos/aislamiento & purificación , Ratas , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Opsinas de Bastones , Transcripción Genética , Triticum/metabolismoRESUMEN
The presence of G-proteins in the eyespot fraction of Chlamydomonas reinhardtii is shown. This fraction is capable of binding (GTP gamma[35S], possesses the GTPase activity and interacts with antibodies raised against a highly conserved peptide of most G-proteins' alpha-subunit. Cross-reaction with a 24-kDa protein is detected on immunoblots. Using an antiserum prepared from vertebrate beta-subunit peptide, two additional proteins with apparent Mr 21 and 29 kDa could be revealed. The light-dependence of GTPase extraction from eyespot membranes is shown. The results make it possible to suggest the participation of G-proteins in the photosensory transduction chain of Ch. reinhardtii.