Quantitative Analysis of RET Receptor Activation and Signaling

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，实现对蛋白质如何执行其功能的定量、机制理解的价值是非常值得赞赏的，并且为这些系统建立了良好的工具和方法。相比之下，我们对另一类具有重要医学意义的蛋白质的功能的定量理解——即被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。在之前的工作中，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关键感觉神经元群体的存活中起重要作用，作为GF受体激活和信号传导定量研究方法的模型系统。RET由四个神经元生长因子家族激活，GDNF、Neurturin、Artemin （ART）和persephin，以及四种膜结合共受体GFR1-4中的一种。激活受体是一种五聚体非共价复合物，由一个生长因子分子结合两个RET分子和两个GFR分子组成。在已发表的工作中，我们已经确定了RET与ART和GFR3一起在活细胞上形成活化受体复合物的步骤序列，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，我们能够建立一个定量的数学模型，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，首次揭示了受体的特定功能特性（如其灵敏度和动态范围）与激活机制的分子细节之间的关系。拟议工作的目标如下：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之外，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预先形成的受体二聚体的变构激活发生的。2. 我们将建立细胞膜上激活的RET受体复合物的组装与细胞信号传导的近端和远端步骤以及细胞存活的功能性细胞反应耦合的定量关系。具体而言，我们将(i)测量RET下游Ras/MAPK、p38MAPK、Akt、Plc/PKC和JNK信号通路中关键信号事件的振幅（激活分子的绝对数量）、演化和衰减动力学以及激活分子状态的固有寿命；（ii）确定每个步骤的哪些信号参数（瞬时振幅、峰值振幅、给定时期内事件的累积数量、生命周期等）对驱动RET刺激下细胞存活反应的振幅和敏感性至关重要；（iii）建立负责从RET激活到细胞存活反应所观察到的渐进式信号敏化的分子机制；（iv）确定不同的信号通路是否与细胞上存在的激活RET水平相似或不同地耦合。3. 我们将比较我们之前建立的ART和GFR3激活RET的机制，以及替代配体/共受体对GDNF/GFR1所利用的机制。我们还将确定激活受体复合物的信号特性是否存在功能上的显著差异。如果成功，这项工作将导致对RET激活和信号传导的机制和定量理解，这对任何其他生长因子受体都是前所未有的，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。

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.