RESUMO
Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT: By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.
Assuntos
Receptores de Vasopressinas , Transdução de Sinais , Arrestinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , LigantesRESUMO
Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through ß-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on ß-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.
Assuntos
Arrestinas/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Transdução de Sinais , Linhagem Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Humanos , Rim/citologia , Rim/embriologia , Rim/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , beta-ArrestinasRESUMO
MOTIVATION: Robustness is the capacity of a system to maintain a function in the face of perturbations. It is essential for the correct functioning of natural and engineered biological systems. Robustness is generally defined in an ad hoc, problem-dependent manner, thus hampering the fruitful development of a theory of biological robustness, recently advocated by Kitano. RESULTS: In this article, we propose a general definition of robustness that applies to any biological function expressible in temporal logic LTL (linear temporal logic), and to broad model classes and perturbation types. Moreover, we propose a computational approach and an implementation in BIOCHAM 2.8 for the automated estimation of the robustness of a given behavior with respect to a given set of perturbations. The applicability and biological relevance of our approach is demonstrated by testing and improving the robustness of the timed behavior of a synthetic transcriptional cascade that could be used as a biological timer for synthetic biology applications. AVAILABILITY: Version 2.8 of BIOCHAM and the transcriptional cascade model are available at http://contraintes.inria.fr/BIOCHAM/.
Assuntos
Biologia Computacional/métodos , Redes Reguladoras de Genes/genética , Algoritmos , Perfilação da Expressão Gênica/métodos , SoftwareRESUMO
G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting.
RESUMO
Vascular endothelial growth factor receptor-2 (VEGFR2) and its ligands (VEGFs) are crucial players in vasculogenesis and angiogenesis. General blocking of this signaling system with antibodies or small molecule inhibitors is an established strategy to treat cancer and age-related macular degeneration. Nevertheless, the activated receptor can signal to discrete downstream signaling pathways and the equilibrium between these pathways is modulated by coreceptors and distinct isoforms of VEGF. Here we investigated the influence of Rab GTPase activating proteins (RabGAPs) on VEGFR2 signaling, tube formation, and migration of endothelial cells. We demonstrate that members of the TBC1D10 subfamily of RabGAPs have opposite effects. Whereas TBC1D10A leads to increased Erk1/2 signaling, TBC1D10B lowered Erk1/2 and p38 signaling and reduced tube formation in vitro. TBC1D10A is a RabGAP acting on RAB13 that was shown before to play a role in angiogenesis and we could indeed show colocalization of these two proteins with VEGFR2 in activated cells. In addition, we observed that cells expressing TBC1D10B show lower expression of VEGFR2 and NRP1 on filopodia of activated cells. Taken together, our systematic analysis of influence of RabGAPs on VEGFR2 signaling identifies the TBC1D10 subfamily members as modulators of angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Neuropilina-1/metabolismo , Suínos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).
Assuntos
Arrestinas/metabolismo , Complexos Multiproteicos/metabolismo , Opsinas/metabolismo , Engenharia de Proteínas/métodos , Rodopsina/metabolismo , Animais , Arrestinas/química , Arrestinas/genética , Bovinos , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Opsinas/química , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Rodopsina/químicaRESUMO
Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.
Assuntos
Baculoviridae/genética , Sistemas CRISPR-Cas , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Transfecção/métodos , Animais , Células COS , Sistemas CRISPR-Cas/genética , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Células Sf9 , Transgenes/genéticaRESUMO
Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst. Squassh3C extends the functionality of Squassh to three fluorescence channels and live-cell movie analysis. SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. It provides segmentation image overview and data exploration, figure generation, object and image filtering, and a statistical significance test in an easy-to-use interface. The overall procedure combines the Squassh3C plug-in for the free biological image processing program ImageJ and a web application working in conjunction with the free statistical environment R, and it is compatible with Linux, MacOS X, or Microsoft Windows. Squassh3C and SquasshAnalyst are available for download at www.psi.ch/lbr/SquasshAnalystEN/SquasshAnalyst.zip.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Filmes Cinematográficos , Imagem Óptica/métodos , Software , Interpretação Estatística de Dados , InternetRESUMO
Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires <1 min and <5 min for 2D and 3D images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.