RESUMO
To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (-)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14â¯ns and 70â¯ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.
Assuntos
Catequina/análogos & derivados , Catequina/farmacologia , Estrogênios/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Sítios de Ligação , Catequina/síntese química , Catequina/química , Bovinos , Cromatografia de Afinidade , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Estrogênios/síntese química , Estrogênios/química , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores de Estrogênio/química , Receptores Acoplados a Proteínas G/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1 h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.
Assuntos
Autorradiografia , Endométrio/citologia , Estradiol/análise , Estradiol/metabolismo , Receptores de Estrogênio/análise , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Animais , Sítios de Ligação , Endométrio/metabolismo , Estradiol/administração & dosagem , Feminino , Imuno-Histoquímica , Camundongos , Gravidez , Receptores de Estrogênio/química , Células Estromais/citologiaRESUMO
PURPOSE: To gain new insight into the roles of cruciferous vegetable-derived bioactive phytochemicals in bone cells, we investigated the effects of indole-3-carbinol (I3C) on cell proliferation and differentiation in estradiol (E2)-exposed calvarial osteoblasts that were obtained from neonatal rats. METHODS: Osteoblast activity was assessed by analyzing cellular DNA, cell-associated osteocalcin (OC) levels and alkaline phosphatase (AP) activity. We also examined [(3)H]-estrone (E1) metabolism and estrogen-agonistic and estrogen-antagonistic activities of 2-hydroxy (OH) E1 and 2-OHE2 and their capacity to displace [(3)H]-E2 at ER binding sites using competition studies. RESULTS: I3C did not affect on cellular DNA, OC levels or AP activity. However, I3C completely inhibited E2-induced increases in cell proliferation and differentiation in neonatal rat osteoblasts. Metabolic studies demonstrated that I3C promoted the conversion of [(3)H]-E1 to 2-OHE1 and 2-OHE2 and those higher rates of conversion (twofold-threefold) were archived when a higher dose of I3C was applied. Proliferation and differentiation studies showed that 2-OHE2 but not 2-OHE1 inhibited E2-induced increases in cell proliferation and differentiation via an ER-mediated mechanism. Likewise, Esr1 was expressed at high level than Esr2. 2-OHE1 showed no activity or affinity for ER. CONCLUSIONS: This study is the first to show that a bioactive compound derived from cruciferous vegetables, I3C, abolishes the E2-mediated stimulation of cell activities including, proliferation and differentiation, in rat osteoblasts and increases the 2-hydroxylation of E1, resulting in the formation of inactive and anti-estrogenic metabolites. These results suggest that in neonatal rat osteoblasts, the anti-estrogenic effect of I3C is mediated by 2-OHE2 through ER-α.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Indóis/farmacologia , Osteoblastos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Anticarcinógenos/farmacologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Estradiol/farmacologia , Feminino , Osteoblastos/citologia , Osteoblastos/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.
Assuntos
Apoptose , Fatores de Transcrição NFATC/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Pontos de Checagem do Ciclo Celular , Transformação Celular Neoplásica , Proteína Ligante Fas/química , Proteína Ligante Fas/metabolismo , Humanos , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/metabolismo , Fatores de Transcrição NFATC/química , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The G-protein coupled receptors (GPCRs) represent the largest superfamily of membrane proteins in charge to pass the cell signaling after binding with their cognate ligands to the cell interior. In breast cancer, a GPCR named GPER1 plays a key role in the process of growth and the proliferation of cancer cells. In a previous study, theoretical methods were applied to construct a model of GPER1, which later was submitted to molecular dynamics (MD) simulations to perform a docking calculation. Based on this preceding work, it is known that GPER1 is sensitive to structural differences in its binding site. However, due to the nature of that past study, conformational changes linked to the ligand binding were not observed. Therefore, in this study, in order to explore the conformational changes coupled to the agonist/antagonist binding, MD simulations of about 0.25µs were performed for the free and bound states, summarizing 0.75µs of MD simulation in total. For the bound states, one agonist (G-1) and antagonist (G-15) were chosen since is widely known that these two molecules cause an impact on GPER1 mobility. Based on the conformational ensemble generated through MD simulations, we found that despite G-1 and G-15 being stabilized by similar map of residues, the structural differences between both ligands impact the hydrogen bond pattern not only at the GPER1 binding site but also along the seven-helix bundle, causing significant differences in the conformational mobility along the extracellular and cytoplasmic domain, and to a lesser degree in the curvatures of helix 2, helix 3 and helix 7 between the free and bound states, which is in agreement with reported literature, and might be linked to microscopic characteristics of the activated-inactivated transition. Furthermore, binding free energy calculations using the MM/GBSA method for the bound states, followed by an alanine scanning analysis allowed us to identify some important residues for the complex stabilization.
Assuntos
Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Benzodioxóis/metabolismo , Sítios de Ligação , Ciclopentanos/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Quinolinas/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.
Assuntos
Benzodioxóis/química , Estradiol/análogos & derivados , Estradiol/química , Quinolinas/química , Receptores de Estrogênio/química , Receptores Acoplados a Proteínas G/química , Tamoxifeno/química , Motivos de Aminoácidos , Sítios de Ligação , Fulvestranto , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , TermodinâmicaRESUMO
Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpha (hERalpha) and beta (hERbeta). Because the levels and relative proportion of hERalpha and hERbeta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hERalpha and hERbeta. Significant statistical coefficients were obtained (hERalpha, q(2) = 0.76; hERbeta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hERalpha and hERbeta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design of novel hER modulators with improved selectivity.
Assuntos
Receptores de Estrogênio/química , Sítios de Ligação , Simulação por Computador , Bases de Dados Factuais , Desenho de Fármacos , Feminino , Humanos , Informática , Ligantes , Estrutura Molecular , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Receptores de Estrogênio/classificação , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , TermodinâmicaRESUMO
The estrogens play important role in the homeostatic maintenance of several target tissues including those in the mammary gland, uterus, bone, cardiovascular system, and brain. Most of estrogen's action is thought to be mediated through its nuclear estrogen receptors, ERalpha and ERbeta, which are members of the nuclear receptor superfamily that act as ligand-induced transcription factors. Acting via its receptors, estrogen also plays an essential role in the development and progression of human breast cancer. The ER and progesterone receptor (PR), which are regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. However, the prognosis of a patient with ER+/PR+ breast cancer can be highly variable and a significant proportion of hormone receptor positive breast cancers does not respond to endocrine therapy. The identification of estrogen receptor target genes may improve our understanding of the role played by estrogens in breast cancer making it possible to better tailor hormone treatments and improve a patient's response to hormonal therapy. In this review, we explore the literature for data regarding the identification of estrogen receptor-regulated genes in breast cancer cell lines and breast tumor biopsies using high throughput technologies such as serial analysis of gene expression (SAGE) and cDNA microarrays.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/genética , Antineoplásicos Hormonais/química , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Estradiol/análogos & derivados , Estradiol/química , Estradiol/farmacologia , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/farmacologia , Receptores de Estrogênio/química , Receptores de Estrogênio/efeitos dos fármacos , Tamoxifeno/química , Tamoxifeno/farmacologiaRESUMO
Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17beta-estradiol (E(2)) and the selective ER modulator raloxifene (RAL)] from the human ERalpha ligand-binding domain in monomeric and dimeric forms. E(2) dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II'). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I'). Remarkably, ER dimerization strongly suppresses Paths II and II' for E(2) dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.
Assuntos
Ligantes , Receptores de Estrogênio/química , Núcleo Celular/metabolismo , Simulação por Computador , Dimerização , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Cloridrato de Raloxifeno/farmacologia , SoftwareRESUMO
The estrogen receptor (ER) is an important drug target for the development of novel therapeutic agents for the treatment of breast cancer. Progress towards the design of more potent and selective ER modulators requires the optimization of multiple ligand-receptor interactions. Comparative molecular field analyses (CoMFA) and hologram quantitative structure-activity relationships (HQSAR) were conducted on a large set of ERalpha modulators. Two training sets containing either 127 or 69 compounds were used to generate QSAR models for in vitro binding affinity and potency, respectively. Significant correlation coefficients (affinity models, CoMFA, r(2)=0.93 and q(2)=0.79; HQSAR, r(2)=0.92 and q(2)=0.71; potency models, CoMFA, r(2)=0.94 and q(2)=0.72; HQSAR, r(2)=0.92 and q(2)=0.74) were obtained, indicating the potential of the models for untested compounds. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. The final QSAR models as well as the information gathered from 3D contour maps should be useful for the design of novel ERalpha modulators having improved affinity and potency.
Assuntos
Ligantes , Relação Quantitativa Estrutura-Atividade , Receptores de Estrogênio/química , Sítios de Ligação , Ligação Competitiva , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Estrogênio/metabolismoRESUMO
The estrogen receptor, ER, is an important biological target whose inhibition is known to be therapeutically relevant in the treatment of postmenopausal osteoporosis. In the present study, two prediction methods (CoMFA and GRIND (Almond)) were used to describe the binding modes of a set of estrogen receptor ligands. The critical alignment step presented in CoMFA was solved by using the information of the molecular descriptors space generated by grid-independent descriptors (GRIND). Then, it was possible to build robust and high predictive models based on the alignment-independent model. Since the structure of estrogen receptor is solved, the results of the present 3D QSAR models, given by the PLS maps based on molecular interaction fields (MIF) were compared to ligand-binding ER domains and showed good agreement.
Assuntos
Relação Quantitativa Estrutura-Atividade , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Sítios de Ligação , Ligação Competitiva , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Simulação por Computador , Estrogênios/química , Estrogênios/metabolismo , Feminino , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Ligantes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/metabolismo , Reprodutibilidade dos Testes , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/metabolismoRESUMO
Ultrastructural and immunocytochemical studies of an intra-nuclear particle, the perichromatin granule (PCG), demonstrated the presence of processed mRNA in this structure. Ovariectomy caused an increase in the number of PCGs in uterine cells and administration of estradiol drastically reduced the nuclear pool of PCGs in 15 min. In vitro studies demonstrated that this depletion was accompanied by an increase of the export of previously synthesized RNA. Similar quantitative changes of the abundance of PCG and of the rate of the export of RNA were found in ventral prostate after orchiectomy and testosterone restitution, as well as in the target cells of FSH, LH, TSH, and ACTH. These results taken together led us to conclude that PCGs constitute an intra-nuclear compartment of a few processed mRNA in equilibrium with transcription and export. This mRNA is rapidly transferred to the cytoplasm by specific hormone signals.
Assuntos
Cromatina/química , Receptores de Estrogênio/química , Receptores de Estrogênio/fisiologia , Animais , Endométrio/química , Feminino , RNA Mensageiro/biossíntese , Ratos , Receptores de Estrogênio/análiseRESUMO
OBJECTIVE: The aim of this work was to analyze the effect of estradiol (E(2)), medroxyprogesterone and the two selective estrogen receptor modulators (SERMs) (tamoxifen (Tam) and raloxifene (Ral)) on the estrogen receptor (ER) conformers profile performed by size exclusion HPLC in relation to hormone dependence of mammary tumors. MATERIALS AND METHODS: Two types of mammary tumors were studied: tumors transplanted in BALB/c mice that are medroxyprogesterone acetate (MPA)-dependent for growth, and tumors induced in Sprague-Dawley rats by intraperitoneal injection of N-nitroso-N-methylurea (NMU). Tumors from mice treated with MPA, E(2), Tam or Ral and NMU-treated rats were analyzed and compared to that of control. RESULTS: The tumor conformer profiles were as follows: control and MPA-treated mice showed only one peak (oligomeric form); E(2)-treated mice also showed only one peak (dimer); Tam-treated mice showed one peak corresponding to a possible proteolytic fragment, and Ral-treated mice showed two peaks (oligomeric and a possible proteolytic fragment). On the other hand, NMU-induced mammary tumors from rats showed three peaks (oligomeric, monomeric and proteolytic). CONCLUSION: Our findings may indicate that SERMs affect the aggregation state of ER and thereby its ability to modulate genomic transcription mechanisms related to growth rate.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Cloridrato de Raloxifeno/farmacologia , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Metilnitrosoureia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Hormônio-Dependentes/metabolismo , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/químicaRESUMO
Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17beta-estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17beta-estradiol binding sites of high affinity (Kd = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 +/- 98 vs. 270 +/- 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17alpha-estradiol and the antiestrogen tamoxifen were significantly less effective than 17beta-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2-fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17beta-estradiol binding analysis. The results of this work are consistent with the existence of novel 17beta-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.
Assuntos
Estradiol/metabolismo , Proteínas de Membrana/metabolismo , Útero/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Fracionamento Celular , Membrana Celular/metabolismo , Reações Cruzadas , Citosol/metabolismo , Estradiol/análogos & derivados , Antagonistas de Estrogênios/metabolismo , Feminino , Ligantes , Proteínas de Membrana/imunologia , Microssomos/metabolismo , Peso Molecular , Concentração Osmolar , Ligação Proteica , Coelhos , Receptores de Estrogênio/química , Receptores de Estrogênio/imunologia , Solubilidade , Estereoisomerismo , TripsinaRESUMO
Desde hace varias décadas se sabe que las hormonas esteroides, como la progesterona y el estradiol, son importantes en la regulación de algunos genes involucrados en los procesos de crecimiento, proliferación y diferenciación de las células de la glándula mamaria en diferentes especies animales y en el humano. La presencia o ausencia del receptor de estrógenos es utilizado en la clínica como un marcador de malignidad, evaluación pronóstica, o como parámetro de decisión para el tratamiento con antiestrógenos de algunos tipos de cáncer dependientes de hormonas en la glándula mamaria. La presente revisión tiene por objeto el mostrar algunos de los avances en el conocimiento de la estructura del receptor y su interacción con protooncogenes y factores de crecimiento. Por último, se hace referencia a la relación que tiene la presencia de este receptor en la fisiopatología del cáncer de mama