Tyrosine Kinases in G Protein Mediated Signaling

描述（由申请人提供）：G蛋白偶联受体（GPCR）药物开发传统上集中于正构激动和拮抗的概念，其中受体结构决定下游信号的性质，配体功效决定其强度。在过去的十年中，“多维功效”和“功能选择性”的新范式已经确定，GPCR通过参与经典的G蛋白依赖性和新的G蛋白独立的信号传导网络来介导生物学效应，并且配体结构可以通过稳定活性受体状态以与天然配体不同的比例来“偏置”信号传导。这种"偏向性激动剂"是具有定性改变GPCR信号传导的能力的新型药理学实体，实际上产生了具有由配体结构驱动的不同功效谱的新受体。有偏见的激动主义的承诺在于这种能力，以产生'混合'的影响，并在本项目的当前任期内的工作， 证明了抑制蛋白途径选择性激动可以在体内产生用常规激动剂或拮抗剂无法获得的潜在有益效果。同时，我们的数据表明，以“非自然”方式激活GPCR可能导致不可预测的下游生物学后果，特别是当使用选择性激活尚未充分表征的抑制蛋白依赖性信号网络的配体时。因此，开发偏向性治疗剂的努力受到无法基于其体外功效预测抑制蛋白偏向性配体的体内作用的阻碍。该提议的中心假设是，体内结果抑制蛋白依赖性信号传导由相对离散和保守的一组生物反应的激活引起，所述生物反应可以使用适合于药物发现研究的基于细胞的测定法在体外进行分析。在下一个学期，我们计划使用最先进的生物物理和生物化学方法，结合在体内表征偏配体作用来测试这一假设。使用分子内FlAsH BRET传感器监测受体激活后抑制蛋白的构象变化，Aim 1将检查决定抑制蛋白的因素。 "构象特征"，并确定抑制蛋白构象与体外受体/配体功效之间的关系。使用多维功效分析和基于SILAC的全细胞磷酸化蛋白质组学信号网络分析，Aim 2将确定体外可获得的偏向配体效应的范围，并测试不同GPCR之间的抑制蛋白依赖性信号网络是否保守。目标3将通过确定抑制蛋白构象、体外配体功效和与体内暴露于抑制蛋白选择性偏向激动剂相关的生物表型之间的关系来"闭合回路"。这些实验将在骨形成的鼠模型中使用偏向的PTH1甲状旁腺激素受体配体。该项目的完成将有助于建立一个合理的框架， 体外配体功效与抑制蛋白途径选择性偏置GPCR配体的体内作用的关系，提供了对开发新的偏置治疗剂至关重要的信息和工具。

DESCRIPTION (provided by applicant): G protein-coupled receptor (GPCR) drug development has traditionally focused on the concepts of orthosteric agonism and antagonism, in which receptor structure determines the nature of the downstream signal and ligand efficacy determines its intensity. Over the past decade, the newer paradigms of 'pluridimensional efficacy' and 'functional selectivity' have established that GPCRs mediate biological effects by engaging both classical G protein-dependent and novel G protein-independent signaling networks, and that ligand structure can 'bias' signaling by stabilizing active receptor states in different proportions than the native ligand. Such 'biased agonists' are novel pharmacological entities with the ability to qualitatively change GPCR signaling, in effect creating 'new receptors with distinct efficacy profiles driven by ligand structure. The promise of biased agonism resides with this ability to engender 'mixed' effects, and work during the current term of this project has demonstrated that arrestin-pathway selective agonism can produce potentially beneficial effects in vivo that are not attainable with conventional agonists or antagonists. At the same time, our data suggest that activating GPCRs in 'unnatural' ways can lead to unpredictable downstream biological consequences, especially when using ligands that selectively activate as-yet poorly characterized arrestin-dependent signaling networks. Efforts to develop biased therapeutics are thus hampered by an inability to predict the in vivo actions of arrestin-biased ligands based on their in vitro efficacy. The central hypothesis of this proposal is that the in vivo consequences arrestin-dependent signaling arises from activation of a relatively discrete and conserved set of biological responses that can be profiled in vitro using cell-based assays that are amenable to drug discovery research. During the next term, we plan to test this hypothesis using state-of-the-art biophysical and biochemical approaches combined with in vivo characterization of biased ligand action. Using intramolecular FlAsH BRET sensors that monitor conformational changes in arrestins following receptor activation, Aim 1 will examine the factors that determine the arrestin 'conformational signature' and determine the relationship between arrestin conformation and receptor/ligand efficacy in vitro. Using multi-dimensional efficacy profiling and SILAC-based whole cell phosphoproteomic analysis of signaling networks, Aim 2 will determine the range of biased ligand effects attainable in vitro and test whether the arrestin-dependent signaling network is conserved between different GPCRs. Aim 3 will then 'close the loop' by determining the relationship between arrestin conformation, in vitro ligand efficacy, and the biological phenotype associated with in vivo exposure to arrestin-selective biased agonists. These experiments will employ biased PTH1 parathyroid hormone receptor ligands in a murine model of bone formation. Completion of this project will help establish a rational framework for relating in vitro ligand efficacy to the in vivo actions of arrestin pathway-selective biased GPCR ligands, providing information and tools that are critical to the development of novel biased therapeutics.