Quantitative Analysis of RET Receptor Activation and Signaling
RET 受体激活和信号转导的定量分析
基本信息
- 批准号:7899606
- 负责人:
- 金额:$ 28.98万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-03-10 至 2014-01-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAfferent NeuronsAffinityAreaAutoimmune DiseasesAutomobile DrivingBehaviorBindingBiological ModelsCell DeathCell Surface ReceptorsCell SurvivalCell membraneCell surfaceCellsCessation of lifeComplexComputer Systems DevelopmentCoupledCytokine ReceptorsDimensionsDimerizationDistalDrug Delivery SystemsEnvironmentEnzymesEquilibriumErlotinibEtanerceptEventEvolutionFamilyG-Protein-Coupled ReceptorsGDNF geneGDNF receptorsGefitinibGlycosylphosphatidylinositolsGrowth FactorGrowth Factor ReceptorsHumiraInvestigational TherapiesIon ChannelKineticsKnowledgeLeadLearningLifeLigand BindingLigandsLinkMAPK8 geneMalignant NeoplasmsMeasuresMediatingMembraneMethodsModelingMolecularNeuronsPeripheral Nervous SystemPharmaceutical PreparationsPhosphorylationPopulationProcessPropertyProteinsPublishingReceptor ActivationReceptor Protein-Tyrosine KinasesReceptor SignalingRoche brand of trastuzumabSignal PathwaySignal TransductionSolutionsSpinal CordStimulusSurfaceSystemTestingTimeTouch sensationVariantWorkavonexcell growthcell growth regulationcomputerized data processingcytokinedesigndimerfunctional outcomesglial cell-line derived neurotrophic factorhuman RIPK1 proteinimprovedinfancyinfliximabintercellular communicationmathematical modelmembermembrane assemblynervous system disorderneuronal growthneurotrophic factorneurturinnovel strategiespersephinprotein functionpublic health relevancereceptorreceptor densityreceptor functionresearch studyresponsetool
项目摘要
DESCRIPTION (provided by applicant): The value of achieving a quantitative, mechanistic understanding of how proteins perform their functions is well appreciated for protein classes such as enzymes, ion channels and G protein-coupled receptors, and good tools and approaches for such work are well established for these systems. In contrast, our quantitative understanding of the function of another medicinally important class of proteins - that is the large class of multi-component receptors that are activated by protein ligands known as cytokines and Growth Factors (GFs) - is at present only rudimentary. In previous work we have studied RET, a receptor tyrosine kinase that is important in sustaining the survival of a key population of sensory neurons in the peripheral nervous system, as a model system for the development and application of methods for the quantitative study of GF receptor activation and signaling. RET is activated by a family of four neuronal growth factors, GDNF, Neurturin, Artemin (ART) and persephin, in conjunction with one of four membrane-bound co-receptors known as GFR1-4. The activated receptor is a pentameric non-covalent complex comprising one molecule of growth factor bound to two molecules of RET plus two molecules of a GFR. In published work we have established the sequence of steps by which RET, in conjunction with ART and GFR3, form an activated receptor complex on live cells, and have determined the equilibrium constants for all steps, including the steps subsequent to initial ligand binding that occur exclusively on the cell membrane. We were thereby able to develop a quantitative mathematical model that relates the distribution of receptor complexes on the cell surface at a given concentration of ART to the affinities of particular steps, revealing for the first time how specific functional properties of the receptor such as its sensitivity and dynamic range relate to the molecular details of the activation mechanism. The objectives of the proposed work are as follows: 1. we will measure how RET phosphorylation responds to variations in the level of available RET present on the cell surface. In addition to being a stringent test of our proposed mechanism and extending our understanding of this process, these experiments also constitute a novel approach to establishing whether receptor activation occurs by ligand induced dimerization versus allosteric activation of preformed receptor dimers. 2. We will establish the quantitative relationships by which assembly of the activated RET receptor complex on the cell membrane is coupled to proximal and distal steps in cell signaling and to the functional cellular response of cell survival. Specifically, we will (i) measure the amplitude (absolute number of molecules activated), the evolution and decay kinetics, and the intrinsic lifetimes of activated molecular states, for key signaling events downstream of RET in the Ras/MAPK, p38MAPK, Akt, Plc/PKC and JNK signaling pathways; (ii) establish which signaling parameters at each step (instantaneous amplitude, peak amplitude, cumulative number of events over a given period, lifetime, etc.) are critical in driving the amplitude and sensitivity of the cell survival response to RET stimulation; (iii) establish the molecular mechanism responsible for the progressive signal sensitization that is observed from RET activation through to the cell survival response; and (iv) determine whether divergent signaling pathways are coupled similarly or differently to the level of activated RET present on the cell. 3. We will compare the mechanism by which ART and GFR3 bring about RET activation, established in our prior work, with the mechanism utilized by the alternative ligand/co-receptor pair GDNF/GFR1. We will additionally determine whether there are functionally significant differences in the signaling properties of the activated receptor complexes that result. If successful, the proposed work will result in a mechanistic and quantitative understanding of RET activation and signaling that is unprecedented for any other growth factor receptor, and will provide methods and approaches that can be applied to a wide range of other multi-component receptor systems.
PUBLIC HEALTH RELEVANCE: The signals that cells send each other to regulate critical functions such as cell growth, maturation, and cell death are primarily mediated by a large and diverse family of messenger proteins known as cytokines and growth factors (GFs). These proteins are secreted into the intracellular medium, and are recognized by cells that possess appropriate 'receptor' proteins on their surface. The receptors become activated upon GF binding, leading to initiation of an intracellular signaling cascade that alters the behavior of the receiving cell in specific ways. Many important drugs, including Enbrel(R), Remicade(R), Humira(R), Herceptin(R), Avonex(R), Tarceva(R), and Iressa(R), treat a variety of cancers, autoimmune diseases and neurological disorders by disrupting GF receptor signaling. The hundreds of GFs and their receptors that have not so far been successfully targeted by approved drugs remain a fertile ground for new experimental therapies. In recent decades we have learned a great deal about the different proteins that function as cytokines or as part of cytokine receptors, and about the combinations in which they interact. However, in comparison with other medicinally important protein classes - such as enzymes, ion channels and G protein-coupled receptors - we still know remarkably little about how GF receptors perform their function. In particular, in almost no case do we understand the quantitative details of how GF binding is coupled to receptor activation, or receptor activation is in turn coupled to intracellular signaling, making it impossible to say, for example, how the properties of a given receptor lead a cell to respond to a particular level of GF in its environment. The proposed work aims to address this important gap in our knowledge by using the GF receptor RET, which is important in sustaining the survival of a key population of nerve cells in the spinal cord, as a model system to elucidate how GF/GF receptor interactions are coupled to intracellular signaling and to the resulting cellular response. This work builds directly upon prior successful work with the RET receptor in the PI's lab. If successful, the new knowledge and experimental methods it will deliver have the potential to contribute new and improved approaches to discovering and developing drugs that target GFs and their receptors.
描述(由申请人提供):对于酶、离子通道和 G 蛋白偶联受体等蛋白质类别,人们充分认识到对蛋白质如何发挥其功能进行定量、机制理解的价值,并且用于此类工作的良好工具和方法是为这些系统建立了良好的基础。相比之下,我们对另一类医学上重要的蛋白质——即被称为细胞因子和生长因子(GF)的蛋白质配体激活的一大类多组分受体——的功能的定量理解目前还只是初步的。在之前的工作中,我们研究了 RET,一种受体酪氨酸激酶,对于维持周围神经系统中关键感觉神经元群体的生存至关重要,作为开发和应用 GF 受体定量研究方法的模型系统激活和信号传导。 RET 由四种神经元生长因子家族(GDNF、Neurturin、Artemin (ART) 和persephin)与四种膜结合共受体之一(称为 GFR1-4)一起激活。激活的受体是五聚体非共价复合物,包含与两分子 RET 结合的一分子生长因子以及两分子 GFR。在已发表的工作中,我们建立了 RET 与 ART 和 GFR3 结合在活细胞上形成活化受体复合物的步骤顺序,并确定了所有步骤的平衡常数,包括发生初始配体结合之后的步骤只存在于细胞膜上。因此,我们能够开发一个定量数学模型,将给定 ART 浓度下细胞表面受体复合物的分布与特定步骤的亲和力联系起来,首次揭示受体的特定功能特性(例如其敏感性)如何影响和动态范围与激活机制的分子细节有关。拟议工作的目标如下: 1. 我们将测量 RET 磷酸化如何响应细胞表面可用 RET 水平的变化。除了对我们提出的机制进行严格测试并扩展我们对该过程的理解之外,这些实验还构成了一种新方法来确定受体激活是否是通过配体诱导的二聚化与预先形成的受体二聚体的变构激活发生的。 2. 我们将建立定量关系,通过该定量关系,细胞膜上激活的 RET 受体复合物的组装与细胞信号传导中的近端和远端步骤以及细胞存活的功能性细胞反应耦合。具体来说,我们将 (i) 测量 Ras/MAPK、p38MAPK、Akt、 Plc/PKC和JNK信号通路; (ii) 确定每个步骤的哪些信号参数(瞬时幅度、峰值幅度、给定时期内的累积事件数、寿命等)对于驱动细胞存活对 RET 刺激的反应的幅度和敏感性至关重要; (iii) 建立负责渐进信号敏化的分子机制,从 RET 激活到细胞存活反应均可观察到; (iv) 确定不同的信号传导途径与细胞上存在的活化 RET 水平的偶联是否相似或不同。 3. 我们将比较我们之前工作中建立的 ART 和 GFR3 引起 RET 激活的机制与替代配体/共受体对 GDNF/GFR1 使用的机制。我们还将确定所产生的激活受体复合物的信号传导特性是否存在功能上的显着差异。如果成功,拟议的工作将导致对 RET 激活和信号传导的机制和定量理解,这对于任何其他生长因子受体来说都是前所未有的,并将提供可广泛应用于其他多组分受体系统的方法和途径。
公共健康相关性:细胞相互发送以调节细胞生长、成熟和细胞死亡等关键功能的信号主要由称为细胞因子和生长因子 (GF) 的大型且多样化的信使蛋白家族介导。这些蛋白质被分泌到细胞内介质中,并被表面具有适当“受体”蛋白质的细胞识别。受体在 GF 结合后被激活,导致细胞内信号级联的启动,以特定方式改变接收细胞的行为。许多重要的药物,包括 Enbrel(R)、Remicade(R)、Humira(R)、Herceptin(R)、Avonex(R)、Tarceva(R) 和 Iressa(R),可治疗多种癌症、自身免疫性疾病和通过破坏 GF 受体信号传导而导致神经系统疾病。迄今为止,尚未成功被批准药物靶向的数百种 GF 及其受体仍然是新实验疗法的沃土。近几十年来,我们对作为细胞因子或细胞因子受体一部分的不同蛋白质以及它们相互作用的组合了解了很多。然而,与其他医学上重要的蛋白质类别(例如酶、离子通道和 G 蛋白偶联受体)相比,我们对 GF 受体如何发挥其功能仍然知之甚少。特别是,在几乎任何情况下,我们都不了解 GF 结合如何与受体激活耦合的定量细节,或者受体激活反过来如何与细胞内信号传导耦合,这使得我们不可能说,例如,给定受体的特性如何引导细胞对其环境中特定水平的 GF 做出反应。拟议的工作旨在通过使用 GF 受体 RET(对于维持脊髓中关键神经细胞群的生存很重要)作为模型系统来阐明 GF/GF 受体如何相互作用,来填补我们知识中的这一重要空白与细胞内信号传导以及由此产生的细胞反应耦合。这项工作直接建立在 PI 实验室之前对 RET 受体的成功研究基础上。如果成功,它将提供的新知识和实验方法有可能为发现和开发针对 GF 及其受体的药物提供新的和改进的方法。
项目成果
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Adrian Whitty其他文献
Adrian Whitty的其他文献
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