ABSTRACT
How receptor tyrosine kinase (RTK) growth signaling is controlled physiologically is incompletely understood. We have previously provided evidence that the survival and mitotic activities of vascular endothelial cell growth factor receptor-2 (VEGFR2) signaling are dependent on C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 joint signaling in a physically interactive platform. Herein, we document that the platelet derived and epidermal growth factor receptors (PDGFR and EGFR) are regulated by the same interconnection and clarify the mechanism underlying the dependence. We show that the joint signaling is required to overcome dominant restraint on RTK function by the combined repression of tonically activated PHLPP, SOCS1/SOCS3, and CK2/Fyn dependent PTEN. Signaling studies showed that augmented PI-3KÉ£ activation is the process that overcomes the multilevel growth restraint. Live-cell flow cytometry and single-particle tracking indicated that blockade of C3ar1/C5ar1 or IL-6R signaling suppresses RTK growth factor binding and RTK complex formation. C3ar1/C5ar1 blockade abrogated growth signaling of four additional RTKs. Active relief of dominant growth repression via joint C3ar1/C5ar1 and IL-6R joint signaling thus enables RTK mitotic/survival signaling.
Subject(s)
Endothelial Cells/metabolism , PTEN Phosphohydrolase/metabolism , Phosphoprotein Phosphatases/metabolism , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Complement/metabolism , Receptors, Interleukin-6/metabolism , Signal Transduction , Suppressor of Cytokine Signaling 1 Protein/metabolism , Suppressor of Cytokine Signaling 3 Protein/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Cell Line , Endothelial Cells/cytology , Genes, Dominant , Mice , Mice, Knockout , PTEN Phosphohydrolase/genetics , Phosphoprotein Phosphatases/genetics , Receptor, Anaphylatoxin C5a/genetics , Receptors, Complement/genetics , Receptors, Interleukin-6/genetics , Suppressor of Cytokine Signaling 1 Protein/genetics , Suppressor of Cytokine Signaling 3 Protein/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
Fc receptors (FcRs) are an important bridge between the innate and adaptive immune system. Fc gamma receptor I (FcγRI; CD64), the high-affinity receptor for immunoglobulin G (IgG), plays roles in inflammation, autoimmune responses, and immunotherapy. Stimulation of myeloid cells with cytokines, such as tumor necrosis factor-α ( TNFα) and interferon-γ ( IFNγ), increases the binding of FcγRI to immune complexes (ICs), such as antibody-opsonized pathogens or tumor cells, through a process known as "inside-out" signaling. Using super-resolution imaging, we found that stimulation of cells with IL-3 also enhanced the clustering of FcγRI both before and after exposure to ICs. This increased clustering was dependent on an intact actin cytoskeleton. We found that chemical inhibition of the activity of the phosphatase PP1 reduced FcγRI inside-out signaling, although the phosphorylation of FcγRI itself was unaffected. Furthermore, the antibody-dependent cytotoxic activity of human neutrophils toward CD20-expressing tumor cells was increased after stimulation with TNFα and IFNγ. These results suggest that nanoscale reorganization of FcγRI, stimulated by cytokine-induced, inside-out signaling, enhances FcγRI cellular effector functions.
Subject(s)
Actin Cytoskeleton/metabolism , Interferon-gamma/pharmacology , Neutrophils/metabolism , Receptors, IgG/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Membrane/metabolism , Cells, Cultured , Humans , Immunoglobulin G/metabolism , Mice , Myeloid Cells/metabolism , Neutrophils/drug effects , Neutrophils/immunology , Phosphorylation , Receptors, IgG/genetics , Signal TransductionABSTRACT
Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF-making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G-protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.
Subject(s)
Epigen/chemistry , Epiregulin/chemistry , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Crystallography, X-Ray , Epigen/metabolism , Epiregulin/metabolism , Fluorescence Resonance Energy Transfer , Humans , Kinetics , Ligands , Models, Molecular , Protein MultimerizationABSTRACT
The challenge of crystallizing single-pass plasma membrane receptors has remained an obstacle to understanding the structural mechanisms that connect extracellular ligand binding to cytosolic activation. For example, the complex interplay between receptor oligomerization and conformational dynamics has been, historically, only inferred from static structures of isolated receptor domains. A fundamental challenge in the field of membrane receptor biology, then, has been to integrate experimentally observable dynamics of full-length receptors (e.g. diffusion and conformational flexibility) into static structural models of the disparate domains. In certain receptor families, e.g. the ErbB receptors, structures have led somewhat linearly to a putative model of activation. In other families, e.g. the tumor necrosis factor (TNF) receptors, structures have produced divergent hypothetical mechanisms of activation and transduction. Here, we discuss in detail these and other related receptors, with the goal of illuminating the current challenges and opportunities in building comprehensive models of single-pass receptor activation. The deepening understanding of these receptors has recently been accelerated by new experimental and computational tools that offer orthogonal perspectives on both structure and dynamics. As such, this review aims to contextualize those technological developments as we highlight the elegant and complex conformational communication between receptor domains. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.
Subject(s)
Cell Membrane/genetics , ErbB Receptors/genetics , Receptors, Tumor Necrosis Factor/genetics , Structure-Activity Relationship , Cell Membrane/chemistry , Cell Membrane/metabolism , Crystallography, X-Ray , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Humans , Models, Molecular , Protein Conformation , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/metabolismABSTRACT
Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix-helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.
Subject(s)
Cholesterol/metabolism , Membrane Lipids/metabolism , Protein Multimerization , Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Caspases/metabolism , Humans , Jurkat Cells , Models, Biological , Protein Conformation , ProteolysisABSTRACT
Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we found that oxidation increased the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction, by 0.5-1.4 kcal/mol. We found that non-hydrogen-bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups were enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR spectroscopy experiments on model peptides demonstrated that oxidation of methionine stabilized the interaction by 0.5-0.6 kcal/mol. We confirmed the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on (i) calmodulin structure and dynamics, and (ii) lymphotoxin-α binding toTNFR1. Thus, the methionine-aromatic motif was a determinant of protein structural and functional sensitivity to oxidative stress.
Subject(s)
Hydrocarbons, Aromatic/chemistry , Methionine/chemistry , Hydrocarbons, Aromatic/metabolism , Methionine/metabolism , Models, Molecular , Oxidation-Reduction , Quantum TheoryABSTRACT
Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non-small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.
Subject(s)
Carcinoma, Non-Small-Cell Lung/enzymology , EGF Family of Proteins/metabolism , ErbB Receptors/metabolism , Lung Neoplasms/enzymology , Animals , CHO Cells , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation , Cell Transformation, Neoplastic , Cricetulus , EGF Family of Proteins/genetics , ErbB Receptors/genetics , HeLa Cells , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Microscopy, Confocal , Mutation , Phosphorylation , Protein Aggregates , Protein Kinase Inhibitors , Protein Multimerization , Signal TransductionABSTRACT
Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.
Subject(s)
Fluorescent Dyes , Microscopy, Fluorescence/methods , Cell Line , Clathrin/metabolism , Clathrin-Coated Vesicles/metabolism , Endocytosis , ErbB Receptors/metabolism , Humans , Protein Binding , Protein Transport , Tubulin/metabolismABSTRACT
Signal transduction is regulated by protein-protein interactions. In the case of the ErbB family of receptor tyrosine kinases (RTKs), the precise nature of these interactions remains a topic of debate. In this review, we describe state-of-the-art imaging techniques that are providing new details into receptor dynamics, clustering, and interactions. We present the general principles of these techniques, their limitations, and the unique observations they provide about ErbB spatiotemporal organization.
Subject(s)
ErbB Receptors/physiology , Image Processing, Computer-Assisted/methods , Microscopy, Electron, Transmission/methods , Microscopy, Fluorescence/methods , Signal Transduction/physiology , ErbB Receptors/ultrastructure , Fluorescence Resonance Energy Transfer/methods , HumansABSTRACT
Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1-1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.
Subject(s)
Lymphotoxin-alpha/chemistry , Methionine/chemistry , Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , Receptors, Tumor Necrosis Factor, Type I/chemistry , TNF-Related Apoptosis-Inducing Ligand/chemistry , Amino Acid Motifs , HEK293 Cells , Humans , Jurkat Cells , Lymphotoxin-alpha/genetics , Lymphotoxin-alpha/metabolism , Methionine/genetics , Methionine/metabolism , Mutation , Protein Stability , Protein Structure, Quaternary , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolismABSTRACT
Apoptosis is one of the core signaling pathways disrupted in pancreatic ductal adenocarcinoma (PDA). Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF)-receptor superfamily that is expressed in cancer cells. Binding of TNF-related apoptosis-inducing ligand (TRAIL) to DR5 is a potent trigger of the extrinsic apoptotic pathway, and numerous clinical trials are based on DR5-targeted therapies for cancer, including PDA. Human antigen R (HuR), an RNA-binding protein, regulates a select number of transcripts under stress conditions. Here we report that HuR translocates from the nucleus to the cytoplasm of PDA cells upon treatment with a DR5 agonist. High doses of DR5 agonist induce cleavage of both HuR and caspase 8. HuR binds to DR5 mRNA at the 5'-untranslated region (UTR) in PDA cells in response to different cancer-associated stressors and subsequently represses DR5 protein expression; silencing HuR augments DR5 protein production by enabling its translation and thus enhances apoptosis. In PDA specimens (n = 53), negative HuR cytoplasmic expression correlated with elevated DR5 expression (odds ratio 16.1, p < 0.0001). Together, these data demonstrate a feedback mechanism elicited by HuR-mediated repression of the key apoptotic membrane protein DR5.
Subject(s)
ELAV Proteins/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , RNA Processing, Post-Transcriptional , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , 5' Untranslated Regions , Antimetabolites, Antineoplastic/pharmacology , Cell Line, Tumor , Cytoplasm/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , ELAV Proteins/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Protein Transport/drug effects , Proteolysis/drug effects , RNA, Messenger , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , GemcitabineABSTRACT
The widely accepted model for tumor necrosis factor 1 (TNFR1) signaling is that ligand binding causes receptor trimerization, which triggers a reorganization of cytosolic domains and thus initiates intracellular signaling. This model of stoichiometrically driven receptor activation does not account for the occurrence of ligand independent signaling in overexpressed systems, nor does it explain the constitutive activity of the R92Q mutant associated with TRAPS. More recently, ligand binding has been shown to result in the formation of high molecular weight, oligomeric networks. Although the dimer, shown to be the preligand structure, is thought to remain present within ligand-receptor networks, it is unknown whether network formation or ligand-induced structural change to the dimer itself is the trigger for TNFR1 signaling. In the present study, we investigate the available crystal structures of TNFR1 to explore backbone dynamics and infer conformational transitions associated with ligand binding. Using normal-mode analysis, we characterize the dynamic coupling between the TNFR1 ligand binding and membrane proximal domains and suggest a mechanism for ligand-induced activation. Furthermore, our data are supported experimentally by FRET showing that the constitutively active R92Q mutant adopts an altered conformation compared to wild-type. Collectively, our results suggest that the signaling competent architecture is the receptor dimer and that ligand binding modifies domain mobilities intrinsic to the receptor structure, allowing it to sample a separate, active conformation mediated by network formation.
Subject(s)
Hereditary Autoinflammatory Diseases/physiopathology , Receptors, Tumor Necrosis Factor, Type I/physiology , Signal Transduction/physiology , Fever , Fluorescence Resonance Energy Transfer , HEK293 Cells , Hereditary Autoinflammatory Diseases/genetics , Humans , Ligands , Models, Molecular , Point Mutation , Protein Binding , Protein Conformation/drug effects , Protein Multimerization , Protein Structure, Tertiary , Receptors, Tumor Necrosis Factor, Type I/geneticsABSTRACT
Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.
Subject(s)
Apoptosis/physiology , Models, Biological , Protein Multimerization/physiology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Jurkat Cells , Microscopy, Fluorescence , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/geneticsABSTRACT
It is generally accepted that ions interact directly with lipids in biological membranes. Decades of biophysical studies on pure lipid bilayer systems have shown that only certain types of ions, most significantly large anions and multivalent cations, can fundamentally alter the structure and dynamics of lipid bilayers. It has long been accepted that at physiological concentrations NaCl ions do not alter the physical behavior or structure of bilayers composed solely of zwitterionic phosphatidylcholine (PC) lipids. Recent X-ray scattering experiments have reaffirmed this dogma, showing that below 1 M concentration, NaCl does not significantly alter bilayer structure. However, despite this history, there is an ongoing controversy within the molecular dynamics (MD) simulation community regarding NaCl/PC interactions. In particular, the CHARMM and GROMOS force fields show dramatically different behavior, including the effect on bilayer structure, surface potential, and the ability to form stable, coordinated ion-lipid complexes. Here, using long-timescale, constant-pressure simulations under the newest version of the CHARMM force field, we find that Na⺠and Cl⻠associate with PC head groups in a POPC bilayer with approximately equal, though weak, affinity, and that the salt has a negligible effect on bilayer structure, consistent with earlier CHARMM results and more recent X-ray data. The results suggest that interpretation of simulations where lipids interact with charged groups of any sort, including charged proteins, must be carefully scrutinized.
Subject(s)
Ions/chemistry , Lipid Bilayers/chemistry , Phosphatidylcholines/chemistry , Sodium Chloride/chemistry , Water/chemistry , Molecular Dynamics SimulationABSTRACT
Estrogen receptor-alpha (ERalpha) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERalpha protein by targeting receptor for destruction by the 26S proteasome. However, ERalpha is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulse-chase analysis was developed using a tet-inducible ERalpha expression system to determine the rate of ERalpha protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERalpha half-life is shortened from 3 to 1 h. However, ERalpha half-life increases over time, achieving a half-life of approximately 6 h in 72 h of estrogen treatment. Analysis of ERalpha half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERalpha and showed that the half-life of the phosphomimetic ERalpha mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERalpha protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.
Subject(s)
Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Protein Isoforms/metabolism , Cell Line, Tumor , Estrogen Receptor alpha/genetics , Humans , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Isoforms/genetics , Time FactorsABSTRACT
Estrogen receptor-alpha (ERalpha) is a transcriptional activator whose concentration is tightly regulated by the cellular environment. In breast tumors of postmenopausal women, elevated receptor concentrations can be associated with negative clinical outcomes, yet it remains poorly understood how such high levels impact ERalpha function. We previously demonstrated that high nuclear concentrations of ERalpha in breast cancer cells bypass the requirement for ligand and are sufficient to activate transcription and accelerate proliferation. Here, we extended those studies and asked whether the transcriptional targets and activation mechanism are similar or different from that of estrogen-stimulated ERalpha. We found that at elevated levels, ERalpha activated, but could not repress, known estrogen-responsive genes. Moreover, the set of activated genes was expanded to include the uterine-restricted target gene, complement component 3. The activation mechanism of ERalpha under these conditions depends both on activation function-1 and residues in the proximal region of the ligand-binding domain. Mutations of aspartate 351 and leucine 372 can inhibit ERalpha transcriptional activity gained at high concentrations and discriminate concentration-inducible ERalpha function from that induced by estrogen. Moreover, we demonstrate that at high levels, ERalpha stimulates transcription without recruiting steroid receptor coactivator-3 and without interference by a Gal4-receptor interaction domain box fusion protein containing LxxLL motifs, further distinguishing this mode of regulation from known activation mechanisms. Together these results demonstrate that the concentration of receptor in breast cancer cells can influence the pattern of target gene expression through a noncanonical activation mechanism.
Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/agonists , Gene Expression Regulation, Neoplastic , Transcription Factors/metabolism , Transcriptional Activation/genetics , Amino Acid Sequence , Amino Acid Substitution , Aspartic Acid/chemistry , Aspartic Acid/genetics , Breast Neoplasms/genetics , Complement C3/genetics , Down-Regulation , Estradiol/pharmacology , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leucine/chemistry , Leucine/genetics , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Transcription Factors/genetics , Up-Regulation , Uterus/metabolismABSTRACT
The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.