RESUMEN

hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Estrógenos/análisis , Proteínas de Xenopus , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Diferenciación Celular , Cromosomas Humanos Par 7/genética , Distroglicanos , Proteínas Ligadas a GPI , Regulación Neoplásica de la Expresión Génica , Humanos , Magaininas , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Mapeo Físico de Cromosoma , Proteínas de Plantas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos

RESUMEN

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.

Asunto(s)

Apoptosis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Angiotensina/metabolismo , Animales , Técnicas de Transferencia de Gen , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Células PC12 , Estructura Terciaria de Proteína , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

OBJECTIVE: To examine the possible crosstalk and the roles of angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). METHODS: Fetal VSMCs were prepared from rat fetal aorta at embryonic day 20. Expression of Ang II receptors was measured by a radioligand binding assay. Apoptotic changes were assessed by caspase 3 activity and chromatin dye staining. Regulation of extracellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determining phosphorylated ERK with Western blot. Ang II receptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by protein tyrosine phosphatase assay. RESULTS: The expression of AT1 and AT2 receptors was approximately 70%: 30% per cell. Serum depletion induced apoptosis in fetal VSMCs and selective AT1 receptor stimulation attenuated the apoptotic changes, whereas selective AT2 receptor activation enhanced apoptosis. Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of the AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Moreover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fetal VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a significant decrease of the AT2 receptor-mediated inhibition of ERK phosphorylation and attenuated the proapoptotic effect of AT2 receptor. CONCLUSION: These results indicate that a crosstalk between AT1 and AT2 receptors regulates the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling.

Asunto(s)

Angiotensina II/metabolismo , Apoptosis , Músculo Liso Vascular/embriología , Proteínas Tirosina Fosfatasas/metabolismo , Receptor Cross-Talk , Receptores de Angiotensina/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Activación Enzimática , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/enzimología , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2

RESUMEN

Fibronectin plays an important role in vascular remodeling. A functional interaction between mechanical stimuli and locally produced vasoactive agents is suggested to be crucial for vascular remodeling. We examined the effect of mechanical stretch on fibronectin gene expression in vascular smooth muscle cells and the role of vascular angiotensin II in the regulation of the fibronectin gene in response to stretch. Cyclic stretch induced an increase in vascular fibronectin mRNA levels that was inhibited by actinomycin D and CV11974, an angiotensin II type 1 receptor antagonist; cycloheximide and PD123319, an angiotensin II type 2 receptor antagonist, did not affect the induction. In transfection experiments, fibronectin promoter activity was stimulated by stretch and inhibited by CV11974 but not by PD123319. DNA-protein binding experiments revealed that cyclic stretch enhanced nuclear binding to the AP-1 site, which was partially supershifted by antibody to c-Jun. Site-directed mutation of the AP-1 site significantly decreased the cyclic stretch-mediated activation of fibronectin promoter. Furthermore, antisense c-jun oligonucleotides decreased the stretch-induced stimulation of the fibronectin promoter activity and the mRNA expression. These results suggest that cyclic stretch stimulates vascular fibronectin gene expression mainly via the activation of AP-1 through the angiotensin II type 1 receptor.

Asunto(s)

Fibronectinas/genética , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , Angiotensina II/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Dactinomicina/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/genética , Receptores de Angiotensina/metabolismo , Sistema Renina-Angiotensina/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Activación Transcripcional

RESUMEN

LXRalpha is a member of a nuclear receptor superfamily that regulates transcription. LXRalpha forms a heterodimer with RXRalpha, another member of this family, to regulate the expression of cholesterol 7alpha-hydroxylase by means of binding to the DR4-type cis-element. Here, we describe a function for LXRalpha as a cAMP-responsive regulator of renin and c-myc gene transcriptions by the interaction with a specific cis-acting DNA element, CNRE (an overlapping cAMP response element and a negative response element). Our previous studies showed that renin gene expression is regulated by cAMP, at least partly, through the CNRE sequence in its 5'-flanking region. This sequence is also found in c-myc and several other genes. Based on our cloning results using the yeast one-hybrid system, we discovered that the mouse homologue of human LXRalpha binds to the CNRE and demonstrated that it binds as a monomer. To define the function of LXRalpha on gene expression, we transfected the renin-producing renal As4.1 cells with LXRalpha expression plasmid. Overexpression of LXRalpha in As4.1 cells confers cAMP inducibility to reporter constructs containing the renin CNRE. After stable transfection of LXRalpha, As4.1 cells show a cAMP-inducible up-regulation of renin mRNA expression. In parallel experiments, we demonstrated that LXRalpha can also bind to the homologous CNRE in the c-myc promoter. cAMP promotes transcription through c-myc/CNRE:LXRalpha interaction in LXRalpha transiently transfected cells and increases c-myc mRNA expression in stably transfected cells. Identification of LXRalpha as a cAMP-responsive nuclear modulator of renin and c-myc expression not only has cardiovascular significance but may have generalized implication in the regulation of gene transcription.

Asunto(s)

AMP Cíclico/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/fisiología , Activación Transcripcional , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Clonación Molecular/métodos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN Complementario , Proteínas de Unión al ADN , Humanos , Receptores X del Hígado , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Renina/genética , Elementos de Respuesta , Saccharomyces cerevisiae , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Transfección

RESUMEN

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Asunto(s)

VIH-1/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Secuencia Conservada , Drosophila/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Unión Proteica/genética , Estructura Secundaria de Proteína , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Quinasas S6 Ribosómicas/química

RESUMEN

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.

Asunto(s)

Genoma Viral , Hepacivirus/genética , Proteínas Virales/metabolismo , Glutatión Transferasa/genética , Hepacivirus/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/genética

RESUMEN

The expression of angiotensin II type 2 (AT(2)) receptor is closely associated with cell growth, differentiation, and/or injury. We examined the effect of interferon (IFN)-gamma on AT(2) receptor expression in mouse fibroblast R3T3 cells and demonstrated that IFN-gamma treatment increased the expression of AT(2) receptor mRNA as well as its binding. Interferon regulatory factor (IRF)-1 was induced in mouse fibroblast R3T3 cells after IFN-gamma stimulation, and electrophoretic mobility shift assay showed an increase in IRF-1 binding with the IRF-specific binding sequence in the AT(2) receptor gene promoter region after IFN-gamma stimulation. The IRF-1 gene promoter contains an IFN-gamma-activated sequence (GAS) motif for possible binding of signal transducer(s) and activator(s) of transcription (STAT). Indeed, in R3T3 cells, IFN-gamma treatment resulted in rapid activation of Janus kinase (Jak) 1, Jak2, and STAT1 via tyrosine phosphorylation. Electrophoretic mobility shift assay with the GAS probe revealed increased STAT1 binding to the IRF-1 gene promoter in response to IFN-gamma stimulation. Transfection of GAS-binding oligonucleotides inhibited the effect of IFN-gamma on IRF-1 production, resulting in the AT(2) receptor trans-activation. Taken together, our data show that IFN-gamma upregulates AT(2) receptor expression in R3T3 cells via the activation of the intracellular Jak/STAT pathway and production of IRF-1.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Interferón gamma/farmacología , Músculo Liso Vascular/enzimología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Angiotensina/genética , Células 3T3/química , Células 3T3/enzimología , Angiotensinas/fisiología , Animales , División Celular/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/inmunología , Factor 1 Regulador del Interferón , Janus Quinasa 1 , Janus Quinasa 2 , Ratones , Músculo Liso Vascular/citología , Oligonucleótidos/farmacología , Regiones Promotoras Genéticas/fisiología , Unión Proteica/genética , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Factor de Transcripción STAT1 , Transducción de Señal/fisiología , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Transfección

RESUMEN

We have identified a novel, membrane-located protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor, which we named ATRAP (for AT1 receptor-associated protein). To further investigate the role of ATRAP in AT1 receptor function, we examined the effect of overexpression of ATRAP on angiotensin II (Ang II)-induced AT1 receptor desensitization and/or internalization, and cell proliferation in adult vascular smooth muscle cells (VSMCs). Transfection of ATRAP potentiated AT1 receptor internalization upon Ang II stimulation in these VSMCs. Moreover, we observed that AT1 receptor-induced DNA synthesis was markedly inhibited in ATRAP transfected VSMCs associated with the inhibition of the phosphorylation of signal transducers and activators of transcription (STAT) 3 and Akt. Our results suggest that ATRAP functions as a negative regulator in AT1 receptor-mediated cell proliferation in VSMCs.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/aislamiento & purificación , Músculo Liso Vascular/citología , Receptores de Angiotensina/metabolismo , Animales , Proteínas Portadoras/fisiología , División Celular/fisiología , Células Cultivadas , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2

RESUMEN

The peptide angiotensin (Ang) II exerts a range of actions in the cardiovascular, renal, reproductive and central nervous systems. At least two distinct Ang II receptor subtypes have been defined and designated as type 1 (AT1) and type 2 (AT2). The function and signaling mechanism of these receptor subtypes are quite different, and these receptors exert opposite effects on cell growth.

RESUMEN

CD46 acts as a cellular receptor for vaccine strains of measles virus (MV). The MV/CD46 interaction-mediated by the MV attachment glycoprotein, the hemagglutinin (H)-not only facilitates infection but also induces CD46 downregulation. A conflict of opinion exists as to whether a single MVH binding site on CD46, or two separate sites, facilitates the two phenomena. To investigate this conundrum we first tested and compared a panel of CD46-specific monoclonal antibodies (mAbs) for their capacity to block both processes. One (mAb 13/42) abrogated both MV fusion and CD46 downregulation. Mutation of an amino acid (arg59 in the SCR1 of CD46) essential for the epitope of mAb 13/42 resulted in the abrogation of both CD46 downregulation and viral fusion. This strongly suggests that the same MV binding site on CD46 is responsible for both CD46 downregulation and MV infection.

Asunto(s)

Antígenos CD/metabolismo , Virus del Sarampión/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/genética , Antígenos CD/inmunología , Arginina/genética , Arginina/metabolismo , Células Cultivadas , Regulación hacia Abajo , Mapeo Epitopo , Células HeLa , Humanos , Sarampión/prevención & control , Sarampión/virología , Vacuna Antisarampión/metabolismo , Proteína Cofactora de Membrana , Fusión de Membrana , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Mutagénesis Sitio-Dirigida , Conejos , Receptores Virales/genética , Receptores Virales/inmunología , Virión/metabolismo

RESUMEN

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.

Asunto(s)

Apoptosis/fisiología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Angiotensina/metabolismo , Alanina/genética , Sustitución de Aminoácidos , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Mutagénesis , Mutación , Células PC12/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Ratas , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal

RESUMEN

The angiotensin II type 2 (AT2) receptor is transiently expressed at late gestation in the fetal vasculature, but its expression rapidly declines after birth. We have previously demonstrated that the expression of this receptor mediates decline in vascular DNA synthesis that occurs at this stage of vascular development. To examine further the role of the AT2 receptor in vasculogenesis, we have focused on the effect of the AT2 receptor on vascular smooth muscle cell (VSMC) differentiation. In this study, we examined the time-dependent expression of differentiation markers for VSMCs in the aorta of wild-type and AT2 receptor-null mice. alpha-Smooth muscle actin was expressed at the early stage of differentiation and exhibited unchanged expression before and after the peak of AT2 receptor expression, which was observed at embryonic day 20, neonatal day 1, and thereafter. No difference in alpha-smooth muscle actin expression was observed between the wild-type and AT2 receptor-null mice. In contrast, the mRNA levels for calponin, expressed in the late stage of VSMC differentiation, were significantly higher in the wild-type mouse aorta as compared with the AT2 receptor-null mice, which correlates with expression of the AT2 receptor. Moreover, the protein levels of calponin and high-molecular-weight caldesmon (h-caldesmon) showed lower expression in the aorta of AT2 receptor knockout mice at 2 and 4 weeks after birth. Taken together, our results suggest that the AT2 receptor promotes vascular differentiation and contributes to vasculogenesis.

Asunto(s)

Aorta/fisiología , Músculo Liso Vascular/fisiología , Receptores de Angiotensina/fisiología , Envejecimiento , Animales , Aorta/embriología , Aorta/crecimiento & desarrollo , Presión Sanguínea , Diferenciación Celular , Cruzamientos Genéticos , Desarrollo Embrionario y Fetal , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Mutantes , Desarrollo de Músculos , Músculo Liso Vascular/embriología , Músculo Liso Vascular/crecimiento & desarrollo , ARN Mensajero/genética , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética

RESUMEN

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.

Asunto(s)

Apoptosis , Receptores de Angiotensina/metabolismo , Esfingolípidos/biosíntesis , Aciltransferasas/análisis , Animales , Ceramidas/metabolismo , Fragmentación del ADN , Endopeptidasas/metabolismo , Activación Enzimática , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células PC12 , Ratas , Receptor de Angiotensina Tipo 2 , Serina C-Palmitoiltransferasa , Transducción de Señal

RESUMEN

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Secuencia de Aminoácidos , Angiotensina II/farmacología , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Clonación Molecular , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Distribución Tisular , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Angiotensin II type 2 (AT2) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT2 receptor in AT2 receptor cDNA-transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT1) receptor-mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1alpha/beta, STAT2, and STAT3 without influence on Janus kinase. AT2 receptor activation also inhibited the tyrosine phosphorylation of STAT1alpha/beta induced by interferon-gamma, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT2 receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT2 receptor. Moreover, AT2 receptor inhibited serine phosphorylation of STAT1alpha and STAT3 via the inhibition of extracellular signal-regulated kinase (ERK) activation. Stimulation of AT2 receptor inhibited the binding of STATs with sis-inducing element in c-fos promoter, resulting in decreased c-fos expression. Taken together, our results suggest that AT2 receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Receptores de Angiotensina/fisiología , Transactivadores/metabolismo , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Ratones , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de Angiotensina/clasificación , Factor de Transcripción STAT2 , Factor de Transcripción STAT3 , Serina/metabolismo , Tirosina/metabolismo

RESUMEN

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced growth in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an antigrowth effect in Agtr2(+) VSMC. Growth of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced growth in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC growth was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater growth responses to platelet-derived growth factor and basic fibroblast growth factor, indicating that Agtr2(-) cells exhibit a generalized exaggerated growth phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both growth and ERK phosphorylation dose-dependently, while the regression lines between growth and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the growth enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the growth phenotype of VSMC via the modulation of ERK cascade.

Asunto(s)

Vasos Sanguíneos/crecimiento & desarrollo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Receptores de Angiotensina/genética , Angiotensina II/metabolismo , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , ADN/biosíntesis , Femenino , Flavonoides/farmacología , Imidazoles/farmacología , Losartán/farmacología , Ratones , Ratones Noqueados , Desarrollo de Músculos , Músculo Liso Vascular/crecimiento & desarrollo , Ácido Ocadaico/farmacología , Fosfoproteínas/análisis , Fosforilación , Unión Proteica , Piridinas/farmacología , Receptores de Angiotensina/metabolismo , Vanadatos/farmacología

RESUMEN

Cellular context is an important determinant for the activity of Tat, the trans-activator of human immunodeficiency virus (HIV). We have investigated HIV-1 promoter expression and trans-activation in Saccharomyces cerevisiae to provide clues about the limiting steps for Tat activity in this organism. A minimal 43-nucleotide HIV promoter (HIV43) has the activity of a weak yeast promoter in the presence or absence of various enhancer binding sites (bs), whereas the entire long terminal repeat is not expressed. None of these constructs could be trans-activated by Tat. Fusion proteins Gal4 binding domain (BD)-Tat48 and Gal4BD-Tat72 are active with different efficiencies on various yeast promoters that have Gal4 bs. They have 70 and 50% of Gal4 wild type activity on hybrid HIV promoters fused to Gal4 bs only in the presence of AP1 bs. This study shows that trans-activation of the HIV-1 promoter by Tat occurs in yeast when Tat is targeted to the promoter and a functional enhancer activity is present. Sp1 function and Tat transfer from the RNA to the promoter are two major elements for in vivo trans-activation of HIV-1 that are defective in S. cerevisiae but can be replaced by functional equivalents.

Asunto(s)

Productos del Gen tat/metabolismo , VIH-1/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Transcripción Genética , Activación Transcripcional , Secuencia de Bases , Replicación del ADN , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH/genética , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fleomicinas/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/virología , Factor de Transcripción Sp1/metabolismo , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Integración Viral , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

CD36 is a cell surface receptor that has been shown to interact with a large variety of ligands including thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes, apoptotic neutrophils, modified low density lipoproteins, anionic phospholipids and long chain fatty acids. A number of these CD36 ligands elicit the transduction of intracellular signals involved in cell activation and internalization of bound ligands. The engagement of CD36 possibly activates three cytosolic protein tyrosine kinases that are presumably associated with the C-terminal cytoplasmic tail of CD36. However, the mechanisms by which CD36 functions in ligand binding and signal transduction are poorly understood. In the present study, a membrane-bound and a truncated soluble form of CD36 were expressed in HeLa cells and analyzed by velocity-gradient centrifugation and chemical cross-linking. We show that membrane CD36 exists predominantly as a monomer but a homodimeric form is also found. In contrast, soluble CD36 sedimented in sucrose gradient as a monomer. However, when incubated with thrombospondin, the membrane form of CD36 predominantly sedimented as a dimer whereas soluble CD36 was monomeric. This study shows that thrombospondin has the ability to induce dimerization of CD36 and may be implicated in the signal transduction capacity of this adhesion molecule.

Asunto(s)

Antígenos CD36/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/farmacología , Antígenos CD36/química , Antígenos CD36/efectos de los fármacos , Dimerización , Células HeLa , Humanos , Trombospondinas

RESUMEN

The human CD36 antigen is a scavenger receptor and a celladhesion molecule expressed by platelets, monocytes and microvascular endothelial cells, among other cell types. It belongs to a new and growing family of integral membrane glycoproteins that recognize a wide range of ligands. CD36 has been implicated in hemostasis, thrombosis, malaria, inflammation, lipid metabolism and atherogenesis. Recently, significant advances in CD36 biology have been reported and new CD36-like proteins have been identified.

Asunto(s)

Antígenos CD36/inmunología , Moléculas de Adhesión Celular/inmunología , Endotelio Vascular/inmunología , Animales , Arteriosclerosis/inmunología , Antígenos CD36/genética , Moléculas de Adhesión Celular/genética , Humanos , Inflamación/inmunología , Metabolismo de los Lípidos , Malaria Falciparum/inmunología , Transcripción Genética