RESUMO
Benzyl alcohol (BnOH) is widely used as a component of foods, cosmetics, household products and medical products. It is generally considered to be safe for human use, however, it has been connected to a number of adverse effects, including hypersensitivity reactions and neonatal deaths. BnOH is a membrane fluidizing agent that can affect membrane protein activity and cellular processes such as ligand binding to cell surface receptors, endocytosis and degradation of lysosomal cargo. In this study, we examined the effects of BnOH on intracellular transport using Shiga toxin (Stx), diphtheria toxin (DT) and ricin. BnOH caused reduced toxicity of all three toxins at BnOH concentrations that cause membrane fluidization. The reduced toxicity of Stx and ricin was mainly due to inhibition of retrograde transport between endosomes and the trans-Golgi network as BnOH had small effects on cell association and endocytosis of ricin and Stx. Strikingly, BnOH also induced a reversible fragmentation of the Golgi apparatus.
Assuntos
Álcool Benzílico/farmacologia , Transporte Biológico/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico/efeitos dos fármacos , Toxina Shiga/metabolismo , Rede trans-Golgi/metabolismoRESUMO
EGF receptor (EGFR) and its signaling have been investigated for many years, but how its different ligands regulate signaling has not been thoroughly explored. When investigating EGFR activation and downstream signaling in HeLa cells using a panel of ligands, we found a ligand-dependent differential activation of EGFR and the signaling pathways Akt, PLCγ and STAT with HB-EGF and BTC being the most potent ligands. All the tested ligands induced full activation of Erk signaling at 1 nM, whereas only HB-EGF and partly BTC and EGF induced strong activation of Akt, STAT3 and PLCγ at this concentration. Interestingly, we also found that the high activation potencies of HB-EGF and BTC could only partially be explained by their binding affinities, and are therefore likely to be regulated by other mechanisms. We thus suggest that the signaling pathways initiated from the EGFR vary depending on the ligands bound in a cell specific manner.
Assuntos
Receptores ErbB/metabolismo , Ligantes , Transdução de Sinais , Ligação Competitiva , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Proteoglicanas de Heparan Sulfato/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Concentração Inibidora 50 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-4/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.
Assuntos
Toxina da Cólera/metabolismo , Complexo de Golgi/metabolismo , Ricina/metabolismo , Toxinas Shiga/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Transporte ProteicoRESUMO
The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.
Assuntos
Clatrina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Amilorida/farmacologia , Animais , Betacelulina , Caveolina 1/metabolismo , Clatrina/genética , Dinaminas/metabolismo , Endocitose , Células HeLa , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Camundongos , Células NIH 3T3 , Pinocitose , RNA Interferente Pequeno/metabolismoRESUMO
ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.
Assuntos
Proteínas ADAM/metabolismo , Clatrina/metabolismo , Endocitose , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/química , Proteína ADAM12 , Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Carcinoma/química , Endossomos/metabolismo , Feminino , Proteína Adaptadora GRB2/análise , Células HEK293 , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Domínios Proteicos Ricos em Prolina , Domínios e Motivos de Interação entre ProteínasRESUMO
The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.
Assuntos
Receptores ErbB/antagonistas & inibidores , Expressão Gênica , Quinazolinas/farmacologia , RNA Mensageiro/metabolismo , Receptor ErbB-3/metabolismo , Neoplasias da Mama , Butadienos/farmacologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Gefitinibe , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Morfolinas/farmacologia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-4 , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.
Assuntos
Vesículas Revestidas por Clatrina/enzimologia , Ativação de Macrófagos/fisiologia , Macrófagos/enzimologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Ligante de CD40/genética , Ligante de CD40/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Endossomos/enzimologia , Endossomos/genética , Exocitose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Glicoproteínas de Membrana/genética , Microglia/enzimologia , NADPH Oxidase 2 , NADPH Oxidases/genética , Ratos , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
The epidermal growth factor receptor (EGFR) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand-induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF)-induced EGFR internalization also required Grb2, p38 MAPK-induced internalization did not. Interestingly, AP-2 knock down blocked p38 MAPK-induced EGFR internalization, but only mildly affected EGF-induced internalization. In line with this, simultaneously mutating two AP-2 interaction sites in EGFR affected p38 MAPK-induced internalization much more than EGF-induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Imunofluorescência , Humanos , Fosforilação , Ligação Proteica/efeitos dos fármacosRESUMO
It is now about 20 years since we first wrote reviews about clathrin-independent endocytosis. The challenge at the time was to convince the reader about its existence. Then the suggestion came up that caveolae might be responsible for the uptake. However, clearly this could not be the case since a large fraction of the clathrin-independent uptake is dynamin-independent. Today, two decades later, the field has developed considerably. New techniques have enabled a detailed analysis of several clathrin-independent endocytic mechanisms, and caveolae have been found to be mostly stable structures having several functions of their own. This article aims at providing a brief update on the importance of clathrin-independent endocytic mechanisms, how the processes are regulated differentially, for instance on the poles of polarized cells, and the challenges in studying them.
Assuntos
Endocitose , Animais , Cavéolas/metabolismo , Fenômenos Fisiológicos Celulares , Polaridade Celular , Dinaminas/metabolismo , HumanosRESUMO
Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associated with azurophile granules in neutrophils indicative of A1AT expression being restricted to the promyelocyte stage. We examined the localization and production of A1AT in healthy donor neutrophils and found A1AT to be a constituent of all granule subtypes and to be released from neutrophils following stimulation. A1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor. Neutrophils from patients with A1AT-deficiency carrying the (PI)ZZ mutation in the A1AT gene appeared structurally and functionally normal, but A1AT produced in leukocytes of these patients lacked the ability to bind proteases efficiently. We conclude that A1AT generation and release from neutrophils add significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema.
Assuntos
Neutrófilos/metabolismo , alfa 1-Antitripsina/biossíntese , Estudos de Casos e Controles , Degranulação Celular/efeitos dos fármacos , Diferenciação Celular , Grânulos Citoplasmáticos/metabolismo , Eosinófilos/enzimologia , Exocitose/efeitos dos fármacos , Genótipo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Técnicas In Vitro , Transplante de Fígado , Transplante de Pulmão , Microscopia Eletrônica de Transmissão , Mutação , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Técnica de Janela Cutânea , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/enzimologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia , Deficiência de alfa 1-Antitripsina/cirurgiaRESUMO
Clathrin-dependent endocytosis is a main entry mechanism for the glycolipid-binding Shiga toxin (Stx), although clathrin-independent pathways are also involved. Binding of Stx to its receptor Gb3 not only is essential for Stx retrograde transport to the endoplasmic reticulum and toxicity but also activates signaling through the tyrosine kinase Syk. We previously described that Syk activity is important for Stx entry, but it remained unclear how this kinase modulates endocytosis of Stx. Here we characterized the effects of Stx and Syk on clathrin-coated pit formation. We found that acute treatment with Stx results in an increase in the number of clathrin-coated profiles as determined by electron microscopy and on the number of structures containing the endocytic AP-2 adaptor at the plasma membrane determined by live-cell spinning disk confocal imaging. These responses to Stx require functional Syk activity. We propose that a signaling pathway mediated by Syk and modulated by Stx leads to an increased number of endocytic clathrin-coated structures, thus providing a possible mechanism by which Stx enhances its own endocytosis.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Toxina Shiga/farmacologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Células HeLa , Humanos , Microscopia Eletrônica , Quinase SykRESUMO
Most novel vaccines against infectious diseases are based on recombinant Ag; however, only few studies have compared Ag-specific immune responses induced by natural infection with that induced by the same Ag in a recombinant form. Here, we studied the epitope recognition pattern of the tuberculosis vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti-tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4+ T-cell specific TB10.4 epitope-pattern, which differed completely from that induced by recombinant TB10.4. This difference was not due to post-translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed that both TB10.4 and BCG were transported to Lamp+-compartments. BCG and TB10.4 however, were directed to different types of Lamp+-compartments in the same APC, which may lead to different epitope recognition patterns. In conclusion, we show that different vectors can induce completely different recognition of the same protein.
Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Epitopos de Linfócito T/imunologia , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/genética , Vacina BCG/farmacocinética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/imunologia , Cruzamentos Genéticos , Feminino , Imunidade Inata , Imunização , Interferon gama/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Subpopulações de Linfócitos T/metabolismo , Tuberculose/imunologia , Vacinas contra a Tuberculose/farmacocinética , Vacinas Sintéticas/imunologiaRESUMO
The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.
Assuntos
Proteínas de Membrana/metabolismo , Ricina/metabolismo , Toxina Shiga/metabolismo , Sequência de Bases , Linhagem Celular , Endocitose , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/genética , Microscopia/métodos , Transporte Proteico , RNA Interferente Pequeno , Ricina/toxicidade , Toxina Shiga/toxicidadeRESUMO
Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-alpha causes receptor recycling. TGF-alpha therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.
Assuntos
Endocitose/fisiologia , Receptores ErbB/metabolismo , Ligantes , Transporte Proteico/fisiologia , Anfirregulina , Animais , Betacelulina , Linhagem Celular , Família de Proteínas EGF , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Receptores ErbB/genética , Glicoproteínas/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador alfa/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismoAssuntos
Extensões da Superfície Celular/metabolismo , Pinocitose , Pseudomonas aeruginosa/fisiologia , Vaccinia virus/fisiologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Apoptose/fisiologia , Extensões da Superfície Celular/microbiologia , Extensões da Superfície Celular/virologia , Interações Hospedeiro-PatógenoRESUMO
ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.
Assuntos
Endocitose/fisiologia , Receptores ErbB/metabolismo , Animais , Regulação para Baixo , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.
Assuntos
Endocitose/fisiologia , Fatores de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Animais , Cavéolas/metabolismo , Clatrina/metabolismo , Células Epiteliais/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Pinocitose/fisiologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1 dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation. Both Rac1 and the GTP exchange factor VAV1 were required as upstream signaling proteins in the formyl-methionyl-leucyl-phenylalanine-induced activation of endogenous PAK1. In contrast, PAK1 mutants had no effect on superoxide generation downstream of FcgammaR signaling during phagocytosis of IgG-immune complexes. We further present evidence that the effect of PAK1 on the respiratory burst is mediated through phosphorylation of p47(Phox), and we show that expression of a p47(Phox) (S303D/S304D/S320D) mutant, which mimics phosphorylation by PAK1, induced basal superoxide generation in vivo. In contrast PAK1 substrates LIMK-1 or RhoGDI are not likely to contribute to the PAK1 effect on NADPH oxidase activation. Collectively, our findings define a VAV1-Rac1-PAK1 signaling axis in mononuclear phagocytes regulating superoxide production in a stimulus-dependent manner.
Assuntos
Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complexo Antígeno-Anticorpo/farmacologia , Carcinógenos/farmacologia , Linhagem Celular , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Quinases Lim/genética , Quinases Lim/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Microglia/citologia , Microglia/metabolismo , Mutação de Sentido Incorreto , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidase 2 , NADPH Oxidases/genética , Neuropeptídeos/genética , Fagócitos/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Receptores de IgG/genética , Receptores de IgG/metabolismo , Explosão Respiratória , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Quinases Ativadas por p21/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.
Assuntos
Regulação para Baixo/genética , Endocitose , Receptor ErbB-2/química , Receptor ErbB-2/genética , Benzoquinonas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Mutantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.