权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
8412754
负责人：
Adrian Whitty
金额：
$ 29.71万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
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 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8412754
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 Targeting Environment Enzymes Equilibrium Erlotinib Etanercept Event Evolution Family G-Protein-Coupled Receptors GDNF gene GDNF receptors Gefitinib Glycosylphosphatidylinositols Growth Factor Growth Factor Receptors 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

项目摘要

PROJECT SUMMARY/ABSTRACT Quantitative Analysis of RET Receptor Activation and Signaling 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 one of four membrane-bound co-receptors known as GFR1-4. The activated receptor is a pentameric noncovalent 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.

项目总结/摘要 RET受体活化和信号传导的定量分析实现蛋白质如何执行其功能的定量，机械理解的价值是很好的对于蛋白质类如酶、离子通道和G蛋白偶联受体，对于这些系统来说，开展这类工作的工具和方法已经确立。相反，我们的量化了解另一类重要的医学蛋白质的功能-这是一大类被称为细胞因子和生长因子（GF）的蛋白质配体激活的多组分受体 - 目前只是初步的。在以前的工作中，我们研究了RET，一种受体酪氨酸激酶，在维持周围神经系统中感觉神经元的关键群体的存活方面是重要的，作为GF受体定量研究方法的开发和应用的模型系统激活和信号传导。RET由四种神经元生长因子，GDNF，Neurturin， Artemin（ART）和Persephin，与四种膜结合共受体之一（称为GFR 1 -4）结合。活化的受体是一种五聚体非共价复合物，包含一个生长因子结合的分子，两个RET分子加上两个GFR分子。在已发表的工作中，我们已经建立了序列 RET与ART和GFR 3结合在活细胞上形成活化的受体复合物的步骤，并确定了所有步骤的平衡常数，包括初始配体之后的步骤仅发生在细胞膜上的结合。因此，我们能够开发一个定量的一个数学模型，该模型将给定条件下细胞表面受体复合物的分布联系起来， ART的浓度与特定步骤的亲和力，首次揭示了特定功能如何受体的性质，例如其灵敏度和动态范围，与受体的分子细节有关。激活机制拟议工作的目标如下： 1.我们将测量RET磷酸化如何响应于可用RET水平的变化，存在于细胞表面。除了是对我们提出的机制的严格测试外，扩展了我们对这一过程的理解，这些实验也构成了一种新的方法，确定受体活化是否通过配体诱导的二聚化与变构活化发生形成的受体二聚体。 2.我们将建立定量关系，通过该定量关系，激活的RET受体的组装细胞膜上的复合物与细胞信号传导中的近端和远端步骤偶联，并与细胞膜上的复合物偶联。细胞存活的功能性细胞反应。具体而言，我们将（i）测量振幅（绝对值活化的分子的数量）、演化和衰变动力学以及活化的分子的固有寿命。分子状态，Ras/MAPK，p38 MAPK，Akt， Plc/PKC和JNK信号通路;（ii）确定每个步骤的信号参数（瞬时振幅、峰值振幅、给定周期内事件的累积数量、寿命，等等）。在驱动对RET刺激的细胞存活反应的幅度和灵敏度方面至关重要; (iii)建立负责进行性信号敏化的分子机制，从RET激活到细胞存活反应观察;以及（iv）确定是否存在差异信号传导途径与细胞上存在的活化RET水平类似或不同地偶联。 3.我们将比较ART和GFR 3引起RET激活的机制，在我们先前的工作中建立，具有替代配体/共受体所利用的机制对GDNF/GFR 1。我们还将确定是否存在功能上的显著差异激活的受体复合物的信号特性。如果成功的话，所提出的工作将导致对RET激活的机制和定量理解和信号传导，这是前所未有的任何其他生长因子受体，并将提供方法和这些方法可以应用于广泛的其他多组分受体系统。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

在功能相关的刺激水平下对受体酪氨酸激酶效应器偶联进行定量分析。

DOI：
10.1074/jbc.m114.602268
发表时间：
2015
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Li,Simin;Bhave,Devayani;Chow,JenniferM;Riera,ThomasV;Schlee,Sandra;Rauch,Simone;Atanasova,Mariya;Cate,RichardL;Whitty,Adrian
通讯作者：
Whitty,Adrian

Binding efficiency of protein-protein complexes.