RESUMO
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.
Assuntos
Epigen/química , Epirregulina/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Cristalografia por Raios X , Epigen/metabolismo , Epirregulina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Ligantes , Modelos Moleculares , Multimerização ProteicaRESUMO
Large-scale screens to identify protein interactions typically underperform with eukaryotic extracellular proteins. In this issue, Özkan et al. report development of a high-throughput assay designed specifically for extracellular proteins that uncovers a wealth of new interactions among three protein superfamilies in Drosophila and sets the stage for more extensive screens.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fibronectinas/metabolismo , Imunoglobulinas/metabolismo , Mapas de Interação de Proteínas , Proteínas/metabolismo , Animais , Proteínas de Repetições Ricas em LeucinaRESUMO
Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/metabolismo , Sequência de Aminoácidos , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Células de Purkinje/citologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinapses/fisiologia , Transmissão SinápticaRESUMO
The epidermal growth factor receptor (EGFR) regulates cell proliferation in many tissues. A new structure of the Drosophila EGFR presented by Alvarado et al. (2010) reveals an asymmetric dimer with the ligand bound to only one subunit. The structure provides a rationale for the receptor's negative cooperativity and necessitates a reconsideration of models for activation of human EGFR.
RESUMO
Small proteins perform a diverse array of functions, from microbial competition, to endocrine signaling, to building biomaterials. Microbial systems that can produce recombinant small proteins enable discovery of new effectors, exploration of sequence activity relationships, and have the potential for in vivo delivery. However, we lack simple systems for controlling small-protein secretion from Gram-negative bacteria. Microcins are small-protein antibiotics secreted by Gram-negative bacteria that inhibit the growth of neighboring microbes. They are exported from the cytosol to the environment in a one-step process through a specific class of type I secretion systems (T1SSs). However, relatively little is known about substrate requirements for small proteins exported through microcin T1SSs. Here, we investigate the prototypic microcin V T1SS from Escherichia coli and show that it can export a remarkably wide range of natural and synthetic small proteins. We demonstrate that secretion is largely independent of the cargo protein's chemical properties and appears to be constrained only by protein length. We show that a varied range of bioactive sequences, including an antibacterial protein, a microbial signaling factor, a protease inhibitor, and a human hormone, can all be secreted and elicit their intended biological effect. Secretion through this system is not limited to E. coli, and we demonstrate its function in additional Gram-negative species that can inhabit the gastrointestinal tract. Our findings uncover the highly promiscuous nature of small-protein export through the microcin V T1SS, which has implications for native-cargo capacity and the use of this system in Gram-negative bacteria for small-protein research and delivery. IMPORTANCE Type I secretion systems for microcin export in Gram-negative bacteria transport small antibacterial proteins from the cytoplasm to the extracellular environment in a single step. In nature, each secretion system is generally paired with a specific small protein. We know little about the export capacity of these transporters and how cargo sequence influences secretion. Here, we investigate the microcin V type I system. Remarkably, our studies show that this system can export small proteins of diverse sequence composition and is only limited by protein length. Furthermore, we demonstrate that a wide range of bioactive small proteins can be secreted and that this system can be used in Gram-negative species that colonize the gastrointestinal tract. These findings expand our understanding of secretion through type I systems and their potential uses in a variety of small-protein applications.
Assuntos
Escherichia coli , Sistemas de Secreção Tipo I , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Bactérias Gram-Negativas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase that mediates cell proliferation and differentiation events during development and maintenance of complex organisms. Formation of specific, ligand-dependent EGFR dimers is a key step in stimulating EGFR signaling, and crystal structures of active, dimeric forms of isolated EGFR extracellular regions and kinase domains have revealed much about how dimer interactions regulate EGFR activity. The nature and role of the transmembrane region in regulating EGFR activity remains less clear, however. Proposed roles for the transmembrane region range from nonspecific but energetically favorable interactions to specific transmembrane dimer conformations being associated with active, inactive, or activity-modulated states of EGFR. To investigate the role of specific transmembrane dimers in modulating EGFR activity we generated thirteen EGFR variants with altered transmembrane sequences designed to favor or disfavor specific types of transmembrane region interactions. We show using FRET microscopy that EGFR transmembrane regions have an intrinsic propensity to associate in mammalian cell membranes that is counteracted by the extracellular region. We show using cell-based assays that each of the EGFR transmembrane variants except the Neu variant, which results in constitutive receptor phosphorylation, is able to autophosphorylate and stimulate phosphorylation of downstream effectors Erk and Akt. Our results indicate that many transmembrane sequences, including polyleucine, are compatible with EGFR activity and provide no evidence for specific transmembrane dimers regulating EGFR function.
Assuntos
Receptores ErbB , Transdução de Sinais , Animais , Fosforilação , Receptores ErbB/metabolismo , Transdução de Sinais/fisiologia , Membrana Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Mamíferos/metabolismoRESUMO
The human epidermal growth factor receptor (EGFR/ERBB1) is a receptor tyrosine kinase (RTK) that forms activated oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, human epidermal growth factor receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes. We observe that both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the kinase region at the active state asymmetric kinase dimer interface do not affect the stability of ligand-independent EGFR oligomers. These results indicate that ligand-independent EGFR oligomers form using interactions that are distinct from the EGFR active state.
Assuntos
Multimerização Proteica , Animais , Células CHO , Cricetulus , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Mutação , Domínios Proteicos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
Receptor Tyrosine Kinases (RTKs) comprise a diverse group of cell-surface receptors that mediate key signaling events during animal development and are frequently activated in cancer. We show here that deletion of the extracellular regions of 10 RTKs representing 7 RTK classes or their substitution with the dimeric immunoglobulin Fc region results in constitutive receptor phosphorylation but fails to result in phosphorylation of downstream signaling effectors Erk or Akt. Conversely, substitution of RTK extracellular regions with the extracellular region of the Epidermal Growth Factor Receptor (EGFR) results in increases in effector phosphorylation in response to EGF. These results indicate that the activation signal generated by the EGFR extracellular region is capable of activating at least seven different RTK classes. Failure of phosphorylated Fc-RTK chimeras or RTKs with deleted extracellular regions to stimulate phosphorylation of downstream effectors indicates that either dimerization and receptor phosphorylation per se are insufficient to activate signaling or constitutive dimerization leads to pathway inhibition.
Assuntos
Receptores ErbB/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Receptores ErbB/genética , Humanos , Fosforilação/genética , Fosforilação/fisiologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.
Assuntos
Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Receptores Patched , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, has a complex etiology. Heterozygous, loss-of-function mutations in the sonic hedgehog (SHH) pathway are associated with HPE. However, mutation carriers display highly variable clinical presentation, leading to an "autosomal dominant with modifier" model, in which the penetrance and expressivity of a predisposing mutation is graded by genetic or environmental modifiers. Such modifiers have not been identified. Boc encodes a SHH coreceptor and is a silent HPE modifier gene in mice. Here, we report the identification of missense BOC variants in HPE patients. Consistent with these alleles functioning as HPE modifiers, individual variant BOC proteins had either loss- or gain-of-function properties in cell-based SHH signaling assays. Therefore, in addition to heterozygous loss-of-function mutations in specific SHH pathway genes and an ill-defined environmental component, our findings identify a third variable in HPE: low-frequency modifier genes, BOC being the first identified.
Assuntos
Genes Modificadores , Holoprosencefalia/genética , Imunoglobulina G/genética , Receptores de Superfície Celular/genética , Animais , Expressão Gênica , Variação Genética , Holoprosencefalia/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismoRESUMO
A general aim of studies of signal transduction is to identify mediators of specific signals, order them into pathways, and understand the nature of interactions between individual components and how these interactions alter pathway behavior. Despite years of intensive study and its central importance to animal development and human health, our understanding of the Hedgehog (Hh) signaling pathway remains riddled with gaps, question marks, assumptions, and poorly understood connections. In particular, understanding how interactions between Hh and Patched (Ptc), a 12-pass integral membrane protein, lead to modulation of the function of Smoothened (Smo), a 7-pass integral membrane protein, has defied standard biochemical characterization. Recent structural and biochemical characterizations of Smoothened domains have begun to unlock this riddle, however, and lay the groundwork for improved cancer therapies.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Hedgehog/genética , Humanos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/químicaRESUMO
The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.
Assuntos
Receptor ErbB-2/metabolismo , Receptor ErbB-4/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Drosophila melanogaster , Receptores ErbB/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/genética , RNA de Cadeia Dupla/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , DNA/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Transporte de RNA , RNA de Cadeia Dupla/químicaRESUMO
S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.
Assuntos
Adenosil-Homocisteinase/metabolismo , Lisina/metabolismo , Acetilação , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1ß that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions.
Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Dimerização , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores ErbB/genética , Humanos , Ligantes , Mutagênese/fisiologia , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-4 , Transdução de Sinais/fisiologiaRESUMO
Förster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of â¼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.
Assuntos
Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Receptor ErbB-2/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Multimerização Proteica , Receptor ErbB-2/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.
Assuntos
Adenosina/análogos & derivados , Imunoconjugados/química , Imunoconjugados/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Fenetilaminas/química , Adenosina/química , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Camundongos , Modelos Moleculares , Conformação ProteicaRESUMO
Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.
Assuntos
Detergentes/química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Hedgehog/química , Glicoproteínas de Membrana/química , Receptor Patched-1/genética , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Animais , Baculoviridae/genética , Proteínas de Drosophila/isolamento & purificação , Camundongos , Receptor Patched-1/química , Receptor Patched-1/isolamento & purificação , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/isolamento & purificação , Células Sf9 , SolubilidadeRESUMO
Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.
Assuntos
Sequência Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cristalografia por Raios X , Drosophila melanogaster/química , Fibronectinas/química , Proteínas Ligadas por GPI , Proteínas Hedgehog/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica/genética , Estrutura Terciária de Proteína , Transdução de Sinais , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismoRESUMO
Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.