RESUMEN
Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.
Asunto(s)
Braquidactilia , Proteína Relacionada con la Hormona Paratiroidea , Humanos , Proteína Relacionada con la Hormona Paratiroidea/genética , Braquidactilia/genética , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Metaloproteasas , Proteínas ADAMRESUMEN
Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation.
Asunto(s)
Vaccinia , Animales , Ligandos , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Mamíferos/metabolismoRESUMEN
Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , Transducción de Señal , Ligandos , Hormona Paratiroidea/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiologíaRESUMEN
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
Asunto(s)
Cristalografía por Rayos X , Receptores Adrenérgicos alfa 1/química , Sitios de Unión , Células HEK293 , Humanos , Ligandos , Lípidos/química , Modelos Moleculares , Quinazolinas/química , Quinazolinas/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Receptores Adrenérgicos alfa 2/químicaRESUMEN
Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.
Asunto(s)
Ingeniería de Proteínas/métodos , Receptores Acoplados a Proteínas G/genética , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Proteínas de Unión al GTP/metabolismo , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Resonancia por Plasmón de Superficie/métodosRESUMEN
Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.
RESUMEN
G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Membrana Dobles de Lípidos , Nanoestructuras/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestructura , Regulación Alostérica , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformación Proteica , Receptores de Neurotensina/química , Transducción de SeñalRESUMEN
The parathyroid hormone 1 receptor (PTH1R) is a major regulator of mineral ion homeostasis and bone metabolism and is thus considered an attractive drug target for the treatment of disorders in calcium metabolism and bone-related diseases such as osteoporosis. PTH1R is a member of the class B of GPCRs, which all share a dynamic multidomain binding mechanism to the peptide hormone. For a long time, these complexes have been recalcitrant to structural studies despite their great therapeutic relevance. Through extensive engineering of both the receptor and the peptide agonist ligand, we were able to determine the first high-resolution structure of a PTH1R-agonist complex. Comparisons of the PTH1R crystal structure with subsequently reported cryo-electron microscopy structures of the same receptor in complex with a G protein, as well as with other class B GPCR structures bound to antagonists, reveal new insights into the two-step activation mechanism of class B GPCRs and extend our understanding of the precise molecular rearrangements during receptor activation.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family.
Asunto(s)
Antagonistas del Receptor de Neuroquinina-1/química , Receptores de Neuroquinina-1/química , Aprepitant/química , Aprepitant/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Antagonistas del Receptor de Neuroquinina-1/farmacología , Piperidinas/química , Piperidinas/farmacología , Estructura Secundaria de Proteína , Piridinas/química , Piridinas/farmacología , Receptores de Neuroquinina-1/aislamiento & purificación , Receptores de Neuroquinina-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Parathyroid hormone 1 receptor (PTH1R) is a class B multidomain G-protein-coupled receptor (GPCR) that controls calcium homeostasis. Two endogenous peptide ligands, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), activate the receptor, and their analogs teriparatide and abaloparatide are used in the clinic to increase bone formation as an effective yet costly treatment for osteoporosis. Activation of PTH1R involves binding of the peptide ligand to the receptor extracellular domain (ECD) and transmembrane domain (TMD), a hallmark of class B GPCRs. Here, we present the crystal structure of human PTH1R in complex with a peptide agonist at 2.5-Å resolution, allowing us to delineate the agonist binding mode for this receptor and revealing molecular details within conserved structural motifs that are critical for class B receptor function. Thus, this study provides structural insight into the function of PTH1R and extends our understanding of this therapeutically important class of GPCRs.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1/química , Secuencia de Aminoácidos , Biomimética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Hormona Paratiroidea/química , Péptidos/metabolismo , Unión ProteicaRESUMEN
Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded receptor, are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis.
Asunto(s)
Evolución Molecular Dirigida/métodos , Pliegue de Proteína , Receptores Acoplados a Proteínas G , Animales , Escherichia coli/genética , Humanos , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Western Blotting , Evolución Molecular Dirigida , Humanos , Microscopía Fluorescente , Receptores Acoplados a Proteínas G/genética , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-1/metabolismo , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9 , SpodopteraRESUMEN
Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.
Asunto(s)
Repetición de Anquirina , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Colorantes de Rosanilina/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
UNLABELLED: Viruses take advantage of host posttranslational modifications for their own benefit. It was recently reported that influenza A virus proteins interact extensively with the host sumoylation system. Thereby, several viral proteins, including NS1 and M1, are sumoylated to facilitate viral replication. However, to what extent sumoylation is exploited by influenza A virus is not fully understood. In this study, we found that influenza A virus nucleoprotein (NP) is a bona fide target of sumoylation in both NP-transfected cells and virus-infected cells. We further found that NP is sumoylated at the two most N-terminal residues, lysines 4 and 7, and that sumoylation at lysine 7 of NP is highly conserved across different influenza A virus subtypes and strains, including the recently emerged human H7N9 virus. While NP stability and polymerase activity are little affected by sumoylation, the NP sumoylation-defective WSN-NPK4,7R virus exhibited early cytoplasmic localization of NP. The growth of the WSN-NPK4,7R virus was highly attenuated compared to that of the wild-type WSN virus, and the lysine residue at position 7 is indispensable for the virus's survival, as illustrated by the rapid emergence of revertant viruses. Thus, sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and for virus growth, illustrating sumoylation as a crucial strategy extensively exploited by influenza A virus for survival in its host. IMPORTANCE: Host posttranslational modifications are heavily targeted by viruses for their own benefit. We and others previously reported that influenza A virus interacts extensively with the host sumoylation system. However, the functional outcomes of viral sumoylation are not fully understood. Here we found that influenza A virus nucleoprotein (NP), an essential component for virus replication, is a new target of SUMO. This is the first study to find that NP from different influenza A viruses, including recently emerged H7N9, is sumoylated at conserved lysine 7. Our data further illustrated that sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and virus growth, indicating that influenza A virus relies deeply on sumoylation to survive in host cells. Strategies to downregulate viral sumoylation could thus be a potential antiviral treatment.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/metabolismo , Virus de la Influenza A/metabolismo , Transporte de Proteínas/genética , Proteínas de Unión al ARN/metabolismo , Sumoilación/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Perros , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Virus de la Influenza A/genética , Lisina/genética , Lisina/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Nucleocápside , Proteínas de Unión al ARN/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization.
Asunto(s)
Evolución Molecular Dirigida , Escherichia coli/genética , Receptores de Neurotensina/genética , Transducción de Señal , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Receptores de Neurotensina/química , Homología de Secuencia de AminoácidoRESUMEN
The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor (GPCR) that mediates the endocrine and paracrine effects of parathyroid hormone and related peptides through the activation of phospholipase Cß-, adenylyl cyclase-, mitogen-activated protein kinase-, and ß-arrestin-initiated signaling pathways. It is currently not clear how specificity among these downstream signaling pathways is achieved. A possible mechanism involves adaptor proteins that affect receptor/effector coupling. In a proteomic screen with the PTHR C terminus, we identified vav2, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as a PTHR-interacting protein. The core domains of vav2 bound to the intracellular domains of the PTHR independent of receptor activation. In addition, vav2 specifically interacted with activated Gα(q) but not with Gα(s) subunits, and it competed with PTHR for coupling to Gα(q). Consistent with its specific interaction with Gα(q), vav2 impaired G(q)-mediated inositol phosphate generation but not G(s)-mediated cAMP generation. This inhibition of G(q) signaling was specific for PTHR signaling, compared with other G(q)-coupled GPCRs. Moreover, the benefit for PTHR-mediated inositol phosphate generation in the absence of vav2 required the ezrin binding domain of Na(+)/H(+)-exchanger regulatory factor 1. Our results show that a RhoA GEF can specifically interact with a GPCR and modulate its G protein signaling specificity.
Asunto(s)
Regulación hacia Abajo/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Proteínas Proto-Oncogénicas c-vav/fisiología , Receptor de Hormona Paratiroídea Tipo 1/fisiología , Transducción de Señal/fisiología , Animales , Unión Competitiva/fisiología , Células COS , Chlorocebus aethiops , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Células HEK293 , Humanos , Inositol/metabolismo , Inositol/farmacología , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.
Asunto(s)
Interacciones Huésped-Patógeno , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Proteína SUMO-1/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Perros , Humanos , Inmunohistoquímica , Subtipo H1N1 del Virus de la Influenza A/genética , Lisina/genética , Lisina/metabolismo , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Unión Proteica , Sumoilación , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genética , VirulenciaRESUMEN
ß-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for ß-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in ß-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na(+)/H(+) exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, ß-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and ß-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, ß-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with ß-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and ß-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing ß-arrestin2 into close proximity to the PTHR, thereby facilitating ß-arrestin2 recruitment after receptor activation.
Asunto(s)
Arrestinas/metabolismo , Complejos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Arrestinas/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Complejos Multiproteicos/genética , Fosfoproteínas/genética , Estructura Cuaternaria de Proteína , Receptor de Hormona Paratiroídea Tipo 1/genética , Transducción de Señal/fisiología , Intercambiadores de Sodio-Hidrógeno/genética , beta-ArrestinasRESUMEN
The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas/análisis , Proteínas/metabolismo , Secuencia de Aminoácidos , Arrestinas/análisis , Arrestinas/química , Arrestinas/metabolismo , Línea Celular , Colorantes Fluorescentes/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas/química , Receptor de Hormona Paratiroídea Tipo 1/análisis , Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , beta-ArrestinasRESUMEN
BACKGROUND: Parathyroid hormone (PTH) is a crucial regulator of calcium homoeostasis in humans. Although it is well known that PTH acts primarily on kidney and bone, the precise cellular and subcellular sites of PTH action have not been visualised in human tissues. METHOD: We developed and characterised a novel anti-peptide antibody to the carboxy-terminal region of the human PTH receptor type 1 (PTHR1). Specificity of the antiserum was demonstrated by i) detection of a broad band migrating at M(r) 85,000-95,000 in western blots of membranes from human kidney and PTHR1-transfected cells; ii) cell surface staining of PTHR1-transfected cells; iii) translocation of PTHR1 receptor immunostaining after agonist exposure; and iv) abolition of tissue immunostaining by preadsorption of the antibody with its immunising peptide. The distribution of PTHR1 receptors was investigated in 320 human tumours and their tissues of origin. RESULTS: In the kidney, PTHR1 receptors were predominantly detected at the basolateral plasma membrane of epithelial cells in the proximal and distal tubules but not in the thin limbs of Henle, collecting ducts or glomeruli. In bone, PTHR1 receptors were detected as discrete plasma membrane staining of osteocytes and osteoblasts, whereas osteoclasts remained unstained. In addition, PTHR1 was found in the gut and in a number of neoplastic tissues including colorectal carcinoma, prostate cancer, renal cell carcinoma and osteosarcoma. CONCLUSION: This is the first localisation of PTHR1 receptors in human tissues at the cellular level. The overexpression of PTHR1 receptors may provide a molecular basis for efficient targeting of human tumours with radiolabelled PTH analogues.