RESUMEN
Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Señalización del Calcio , Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genéticaRESUMEN
A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.
Asunto(s)
Neoplasias de la Mama , Calcio , Neoplasias de la Mama/genética , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio de la Dieta , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.
Asunto(s)
Desarrollo Embrionario , Glucocorticoides/farmacología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Empalme del ARN/genética , Activación Transcripcional/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Embrión no Mamífero , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/genética , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Empalme del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transactivadores/fisiología , Activación Transcripcional/efectos de los fármacos , Pez CebraRESUMEN
Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.
Asunto(s)
Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal , Canales Catiónicos TRPV/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcio/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Sulfonamidas/farmacología , Análisis de Supervivencia , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genéticaRESUMEN
The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(ß-TrCP1) and SCF(ß-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(ß-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(ß-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ß-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ß-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.
Asunto(s)
Biotina/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Membrana Nuclear/metabolismo , Fosforilación , Estabilidad Proteica , Reproducibilidad de los Resultados , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECT-domain-containing ubiquitin ligase, promotes the recycling of the ß2-adrenergic receptor (ß2AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the ß2AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo ß2AR-HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the ß2AR. Rab11a, but not Rab11a-K145R, was activated by ß2AR-HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of ß2AR-HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a G-protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase.
Asunto(s)
Receptores Adrenérgicos beta 2/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión al GTP rab/genética , Secuencia de Aminoácidos , Arginina/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Transporte de Proteínas , Receptores Adrenérgicos beta 2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic ß cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 ß cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in ß cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor-PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of ß cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Glucagón/metabolismo , Receptores de Progesterona/metabolismo , Inhibidores de Adenilato Ciclasa , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Secreción de Insulina , Espectrometría de Masas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Receptores de Progesterona/antagonistas & inhibidores , Receptores de Progesterona/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells.
Asunto(s)
Neoplasias de la Mama , Células Endoteliales , Canales Iónicos , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcio/metabolismo , Células Endoteliales/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Plasticidad de la Célula/genéticaRESUMEN
Apoptosis is a highly complex and regulated cell death pathway that safeguards the physiological balance between life and death. Over the past decade, the role of Ca2+ signalling in apoptosis and the mechanisms involved have become clearer. The initiation and execution of apoptosis is coordinated by three distinct groups of cysteines proteases: the caspase, calpain and cathepsin families. Beyond its physiological importance, the ability to evade apoptosis is a prominent hallmark of cancer cells. In this review, we will explore the involvement of Ca2+ in the regulation of caspase, calpain and cathepsin activity, and how the actions of these cysteine proteases alter intracellular Ca2+ handling during apoptosis. We will also explore how apoptosis resistance can be achieved in cancer cells through deregulation of cysteine proteases and remodelling of the Ca2+ signalling toolkit.
Asunto(s)
Apoptosis , Señalización del Calcio , Neoplasias , Humanos , Animales , Neoplasias/metabolismo , Neoplasias/patología , Activación Enzimática , Proteasas de Cisteína/metabolismoRESUMEN
Both matrix stiffening and remodeling of calcium signaling occur in breast cancers, with downstream consequences linked to the progression of the disease. However, the potential intersection between calcium signaling and matrix stiffness has not been fully assessed in models of cancer. Here, we describe the assessment of calcium signaling in breast cancer cells at high and low matrix stiffness using novel gel culture models (gelatin methacryloyl and polydimethylsiloxane) and MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m. Remodeling of ATP-stimulated cytosolic calcium responses in cells on different matrices was assessed using a high throughput fluorescence imaging plate reader. Our data reveal that matrices of higher stiffness attenuate ATP-induced sustained calcium influx in MDA-MB-231 breast cancer cells. This matrix-mediated attenuation of sustained calcium influx was dependent on the store-operated calcium channel component ORAI1. These studies suggest that calcium signaling in breast cancer cells can be altered as a consequence of matrix stiffness; modulation of such pathways may represent a new mechanism to target calcium signaling to regulate tumor progression in breast cancer.
Asunto(s)
Neoplasias de la Mama , Calcio , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Femenino , Gelatina , Humanos , Metacrilatos , Proteína ORAI1/metabolismoRESUMEN
Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.
RESUMEN
The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.
Asunto(s)
Retículo Endoplásmico/metabolismo , Leucina/análisis , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Dimerización , Retículo Endoplásmico/química , Isoleucina/análisis , Isoleucina/metabolismo , Leucina/metabolismo , Fenilalanina/análisis , Fenilalanina/metabolismo , Transporte de Proteínas/fisiología , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Adrenérgicos/análisis , Receptores Adrenérgicos/metabolismo , Receptores Acoplados a Proteínas G/análisis , Transducción de SeñalRESUMEN
Cancer-associated fibroblasts (CAFs) represent an important component of the tumour microenvironment and are implicated in disease progression. Two outstanding questions in cancer biology are how CAFs arise and how they might be targeted therapeutically. The calcium signal also has an important role in tumorigenesis. To date, the role of calcium signalling pathways in the induction of the CAF phenotype remains unexplored. A CAF model was generated through exogenous transforming growth factor beta 1 (TGFß1) stimulation of the normal human mammary fibroblast cell line, HMF3S (HMF3S-CAF), and changes in calcium signalling were investigated. Functional changes in HMF3S-CAF calcium signalling pathways were assessed using a fluorescent indicator, gene expression, gene-silencing and pharmacological approaches. HMF3S-CAF cells demonstrated functionally altered calcium influx pathways with reduced store-operated calcium entry. In support of a calcium signalling switch, two voltage-gated calcium channel (VGCC) family members, CaV1.2 and CaV3.2, were upregulated in HMF3S-CAFs and a subset of patient-derived breast CAFs. Both siRNA-mediated silencing and pharmacological inhibition of CaV1.2 or CaV3.2 significantly impaired CAF activation in HMF3S cells. Our findings show that VGCCs contribute to TGFß1-mediated induction of HMF3S-CAF cells and both transcriptional interference and pharmacological antagonism of CaV1.2 and CaV3.2 inhibit CAF induction. This suggests a potential therapeutic role for targeting calcium signalling in breast CAFs.
RESUMEN
Neuronal calcium sensor-1 (NCS-1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS-1 and breast cancer molecular subtypes and the effects of NCS-1 silencing on calcium (Ca2+ ) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS-1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA-MB-231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS-1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS-1 silencing suppressed unstimulated basal Ca2+ influx. NCS-1 silencing in MDA-MB-231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS-1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store-operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS-1 in clinical breast cancer samples. This newly identified association between NCS-1 and basal breast cancers, together with the identification of the role of NCS-1 in the regulation of the effects of doxorubicin in MDA-MB-231 breast cancer cells, suggests that NCS-1 and/or pathways regulated by NCS-1 may be important in the treatment of basal breast cancers in women.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Muerte Celular/genética , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Adenosina Trifosfato/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Bases de Datos Genéticas , Retículo Endoplásmico/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Necrosis/genética , Necrosis/metabolismo , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , ARN Interferente Pequeño , RNA-Seq , Regulación hacia ArribaRESUMEN
The physiological function of angiotensin II (Ang II) is mediated through the Ang II type 1 (AT1R) and type 2 (AT2R) receptors. Our previous studies have demonstrated that cell surface targeting of AT1R is regulated by Rab and Sar1 GTPases and the F(x)(6)LL motif in the membrane-proximal C terminus. However, the molecular mechanisms underlying the export of nascent AT2R remain poorly defined. In this report, we determined the role of Rab1 GTPase, which specifically controls protein transport from the endoplasmic reticulum (ER) to the Golgi, and receptor dimerization in the biosynthesis of AT2R. Cell surface expression of AT2R was augmented by transient expression of Rab1 and attenuated by dominant-negative Rab1 mutants and small interfering RNA-mediated knockdown of Rab1. Consistently, AT2R inhibition of epidermal growth factor-activated extracellular signal-regulated kinase 1/2 was significantly reduced by the Rab1 mutants, indicating that endogenous Rab1 modulates the cell surface targeting and signaling of AT2R. It is of interest to note that Rab1 augmented the overall expression of AT2R and its mRNA, whereas the Rab1 mutants attenuated the total AT2R expression and enhanced ubiquitin-dependent AT2R degradation. Furthermore, our previously characterized ER export-deficient AT1R mutant in which the F(x)(6)LL motif was mutated formed both homodimers and heterodimers with AT2R. Dimerization of the AT1R mutant with AT2R blocked AT2R trafficking to the cell surface, suggesting constitutive dimerization of both receptors in the ER and an important role of dimerization in ER export of the receptors. These data demonstrate for the first time that Rab1 GTPase and dimerization modulate export traffic from the ER to the cell surface of newly synthesized AT2R.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de la Membrana/fisiología , Multimerización de Proteína/fisiología , Receptor de Angiotensina Tipo 2/biosíntesis , Proteínas de Unión al GTP rab1/fisiología , Línea Celular , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Multimerización de Proteína/genética , Receptor de Angiotensina Tipo 2/genética , Receptor de Angiotensina Tipo 2/metabolismo , Receptor de Angiotensina Tipo 2/fisiología , Proteínas de Unión al GTP rab1/biosíntesis , Proteínas de Unión al GTP rab1/genéticaRESUMEN
A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Complejos Multiproteicos/análisis , Proteínas Recombinantes de Fusión/análisis , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bioensayo/métodos , Línea Celular Transformada , Línea Celular Tumoral , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Luciferasas/análisis , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Plásmidos , Mapeo de Interacción de Proteínas/métodos , Multimerización de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/efectos de la radiación , Proyectos de Investigación , Espectrometría de Fluorescencia/métodos , TransfecciónRESUMEN
Extracellular signals received by G protein-coupled receptors (GPCRs) are transduced into intracellular responses following the activation of heterotrimeric G proteins. As their names suggests, they are composed of three subunits, Galpha and Gbetagamma, the latter being effectively treated as a single entity. The Gbetagamma dimer is assembled with the aid of a number of molecular chaperones in a tightly regulated process. The folding of nascent Gbeta1 is favoured by cellular chaperones such as PhLP-1 and CCT and the ER-resident protein DRiP78 plays an important role in the stability of nascent Ggamma2 subunits. However, much work remains to be done to completely understand the mechanisms underlying assembly of the heterotrimer.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Chaperonas Moleculares/metabolismoRESUMEN
The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells; however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1αï©. The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival; basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.
RESUMEN
Alterations in Ca2+ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca2+ and specific components of Ca2+ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca2+ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca2+ ([Ca2+]CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca2+]CYT. Where cell death occurred, [Ca2+]CYT increases preceded cell death. The sustained increases in [Ca2+]CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca2+ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca2+]CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca2+ indicators for investigating [Ca2+]CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.