Quantitative Analysis of RET Receptor Activation and Signaling

RET 受体激活和信号转导的定量分析

• 批准号: 8040986
• 负责人: Adrian Whitty
• 金额: $ 30.68万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/8040986
• 关键词: Address; Afferent Neurons; Affinity; Area; Autoimmune Diseases; Automobile Driving; Behavior; Binding; Biological Models; Cell Death; Cell Surface Receptors; Cell Survival; Cell membrane; Cell surface; Cells; Cessation of life; Complex; Coupled; Cytokine Receptors; Dimensions; Dimerization; Distal; Drug Delivery Systems; Environment; Enzymes; Equilibrium; Erlotinib; Etanercept; Event; Evolution; Family; G-Protein-Coupled Receptors; GDNF gene; GDNF receptors; Gefitinib; Glycosylphosphatidylinositols; Growth Factor; Growth Factor Receptors; Health; Humira; Investigational Therapies; Ion Channel; Kinetics; Knowledge; Lead; Learning; Life; Ligand Binding; Ligands; Link; MAP Kinase Gene; MAPK8 gene; Malignant Neoplasms; Measures; Mediating; Membrane; Methods; Modeling; Molecular; Neurons; Peripheral Nervous System; Pharmaceutical Preparations; Phosphorylation; Population; Process; Property; Proteins; Publishing; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Roche brand of trastuzumab; Signal Pathway; Signal Transduction; Solutions; Spinal Cord; Stimulus; Surface; System; Systems Development; Testing; Time; Touch sensation; Variant; Work; avonex; cell growth; cell growth regulation; computerized data processing; cytokine; design; dimer; functional outcomes; glial cell-line derived neurotrophic factor; human RIPK1 protein; improved; infancy; infliximab; intercellular communication; mathematical model; member; membrane assembly; nervous system disorder; neuronal growth; neurotrophic factor; neurturin; novel strategies; persephin; protein function; receptor; receptor density; receptor function; research study; response; tool

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.

描述(申请人提供)：对于蛋白质类别，如酶、离子通道和G蛋白偶联受体，实现对蛋白质如何执行其功能的定量、机械性理解的价值被很好地认识到，并且为这些系统很好地建立了用于这类工作的良好工具和方法。相比之下，我们对另一类医学上重要的蛋白质的功能的定量了解--即由称为细胞因子和生长因子(GFS)的蛋白质配体激活的大类多组分受体--目前仅处于初级阶段。在以前的工作中，我们已经研究了RET，一种受体酪氨酸激酶，它对于维持周围神经系统中关键感觉神经元的生存非常重要，作为开发和应用定量研究GF受体激活和信号的方法的模型系统。RET由四种神经生长因子家族激活，即GDNF、Neurturin、Artemin(ART)和Persephin，与四种膜结合的辅助受体之一GFR1-4结合。激活的受体是一种五聚体非共价复合体，由一个生长因子分子与两个RET分子结合，以及两个GFR分子组成。在已发表的工作中，我们建立了RET与ART和GFR3一起在活细胞上形成激活的受体复合体的步骤序列，并确定了所有步骤的平衡常数，包括仅发生在细胞膜上的初始配体结合之后的步骤。因此，我们能够开发一个定量的数学模型，将给定ART浓度下细胞表面受体复合体的分布与特定步骤的亲和力联系起来，首次揭示了受体的特定功能属性(如其敏感性和动态范围)如何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的测试并扩展我们对这一过程的理解外，这些实验还构成了一种新的方法来确定受体激活是通过配体诱导的二聚化还是通过变构激活预先形成的受体二聚体发生的。2.我们将建立激活的RET受体复合体在细胞膜上的组装与细胞信号的近端和远端步骤以及细胞存活的功能性反应之间的定量关系。具体地说，我们将(I)测量在Ras/MAPK、p38MAPK、Akt、Plc/PKC和JNK信号通路中RET下游关键信号事件的振幅(激活的分子绝对数)、演化和衰减动力学以及激活的分子状态的内在寿命；(Ii)确定每个步骤的信号参数(瞬时振幅、峰值振幅、给定时期内事件的累积数量、寿命等)。在驱动细胞对RET刺激的生存反应的幅度和敏感性方面是至关重要的；(Iii)建立负责从RET激活到细胞生存反应的渐进信号敏化的分子机制；以及(Iv)确定不同的信号通路与细胞上激活的RET水平是相似还是不同。3.我们将比较我们先前工作中建立的ART和GFR3导致RET激活的机制，以及替代配体/共受体对GDNF/GFR1所利用的机制。此外，我们还将确定由此产生的激活受体复合体的信号特性是否存在显著差异。如果成功，这项拟议的工作将导致对RET激活和信号的机制和定量的理解，这对任何其他生长因子受体来说都是前所未有的，并将提供可应用于广泛的其他多组分受体系统的方法和途径。