Quantitative Analysis of RET Receptor Activation and Signaling

描述（由申请人提供）：对于蛋白质类（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、机械理解的价值得到了很好的理解，并且对于这些系统，已经很好地建立了用于此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理解-这是一大类多组分受体，被称为细胞因子和生长因子（GF）的蛋白质配体激活-目前只是初步的。在以前的工作中，我们已经研究了RET，受体酪氨酸激酶，这是重要的，在维持周围神经系统中的感觉神经元的关键人口的生存，作为一个模型系统的发展和应用的方法定量研究GF受体的激活和信号。RET由四种神经元生长因子（GDNF、Neurturin、Artemin（ART）和persephin）的家族与被称为GFR 1 -4的四种膜结合共受体之一结合激活。激活的受体是一种五聚体非共价复合物，包含一个生长因子分子与两个RET分子和两个GFR分子结合。在已发表的工作中，我们已经建立了RET与ART和GFR 3结合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，包括仅在细胞膜上发生的初始配体结合之后的步骤。因此，我们能够开发一种定量数学模型，该模型将特定浓度的ART下细胞表面上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。本文的工作目标如下：1.我们将测量RET磷酸化如何响应细胞表面存在的可用RET水平的变化。除了作为我们提出的机制的严格测试和扩展我们对该过程的理解之外，这些实验还构成了一种新的方法来确定受体活化是否通过配体诱导的二聚化与预形成的受体二聚体的变构活化而发生。2.我们将建立的定量关系，通过该组件的激活RET受体复合物的细胞膜上耦合到近端和远端步骤的细胞信号传导和细胞存活的功能性细胞反应。具体而言，我们将（i）测量振幅Ras/MAPK、p38 MAPK、Akt、Plc/PKC和JNK信号传导途径中RET下游关键信号传导事件的活化分子的绝对数量、进化和衰变动力学以及活化分子状态的固有寿命;（ii）确定在每个步骤处的哪些信令参数（瞬时幅度、峰值幅度、在给定时段内的事件的累积数量、寿命等）在驱动对RET刺激的细胞存活应答的幅度和敏感性方面是关键的;（iii）建立负责从RET活化直至细胞存活应答观察到的渐进信号敏化的分子机制;和（iv）确定不同的信号传导途径是否类似地或不同地与细胞上存在的活化RET水平偶联。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）与替代配体/共受体对GDNF/GFR 1所利用的机制。我们还将确定是否有功能上的显着差异，导致激活的受体复合物的信号传导特性。如果成功，拟议的工作将导致对RET激活和信号传导的机械和定量理解，这对于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.