Quantitative Analysis of RET Receptor Activation and Signaling

项目摘要/摘要 RET受体激活及信号转导的定量分析 实现对蛋白质如何执行其功能的定量的、机械性的理解的价值是很好的 对酶、离子通道和G蛋白偶联受体等蛋白质类的赞赏，以及良好的 为这些系统建立了用于此类工作的工具和方法。相比之下，我们的数量 了解另一类在医学上很重要的蛋白质的功能--即一大类蛋白质 被称为细胞因子和生长因子(GFS)的蛋白质配体激活的多组分受体 --目前只是初级阶段。在之前的工作中，我们研究了RET，一种受体酪氨酸激酶，是一种 对于维持周围神经系统中的关键感觉神经元群体的生存非常重要， 作为生长因子受体定量研究方法开发和应用的模型系统 激活和发送信号。RET是由四个神经生长因子家族激活的，GDNF，Neurturin， 青蒿素(ART)和Persephin，以及四种膜结合的共同受体之一，称为GFR1-4。 激活的受体是由一个结合生长因子的分子组成的五聚体非共价复合体。 两个RET分子加上两个GFR分子。在已发表的著作中，我们已经确定了顺序 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， 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，我们能够开发出一种量化的 与给定条件下细胞表面受体复合体分布有关的数学模型 将艺术集中在特定步骤的亲和力上，首次揭示了特定的功能 受体的性质，如其敏感性和动态范围，与分子细节有关 激活机制。拟议工作的目标如下： 1.我们将测量RET磷酸化如何响应可用RET水平的变化 存在于细胞表面。除了是对我们提议的机制和 扩大我们对这一过程的理解，这些实验也构成了一种新的方法 确定受体激活是通过配体诱导的二聚化还是通过变构激活发生的 预先形成的受体二聚体。 2.我们将建立激活的RET受体组装的定量关系 细胞膜上的复合体与细胞信号的近端和远端的步骤以及与 细胞存活的功能性细胞反应。具体地说，我们将(I)测量振幅(绝对 被激活的分子的数量)、演化和衰变动力学以及被激活的固有寿命 Ras/MAPK，p38MAPK，Akt，RET下游关键信号事件的分子状态， PLC/PKC和JNK信号通路；(Ii)确定每个步骤的信号参数 (瞬时幅度、峰值幅度、给定时间段内的累计事件数、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起关键作用； (Iii)建立负责渐进信号敏化的分子机制，即 观察从RET激活到细胞存活反应；以及(Iv)确定是否分化 信号通路与细胞上激活的RET水平相似或不同。 3.我们将比较ART和GFR3激活RET的机制， 在我们之前的工作中建立了，其机制由替代配体/共受体利用 将GDNF/GFR1配对。此外，我们还将确定是否存在功能上的显著差异 在激活的受体复合体的信号特性中.. 如果成功，拟议的工作将导致对RET激活的机械性和量化的理解 和信号，这是前所未有的任何其他生长因子受体，并将提供方法和 可广泛应用于其他多组分受体系统的方法。

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.