Signaling circuits that drive cell movement and ligand scavenging by chemokine receptor CCR2

炎症趋化因子CCL 2及其G蛋白偶联受体介导的单核细胞向组织迁移 CCR 2是正常生理反应和许多疾病包括神经退行性疾病的关键， 创伤性脑损伤、关节炎、糖尿病肾病和非酒精性脂肪肝。此外，CCR 2是 目前正在进行的糖尿病肾病和胰腺癌临床试验的目标。鉴于 尽管CCR 2具有显著的治疗意义，但令人惊讶的是，其在单核细胞功能中的作用知之甚少。在 特别是，虽然CCR 2被公认为促进细胞运动，但很大程度上未被认识到的是，它具有 第二个同样重要的功能，它从细胞外介质中清除趋化因子， 内化并运输趋化因子至溶酶体以进行降解。清除可能使受体- 表达细胞以使其沿着趋化因子浓度增加的梯度移动而不脱敏;它可以 还通过改变特异性受体的可用性来调节不同细胞上受体的反应性。 趋化因子，从而调节是否以及什么类型的细胞迁移。我们假设，事实上， 这两种功能（细胞运动和清除）的内聚整合使CCR 2能够有效地 在不同的生物环境中控制定向细胞迁移。此外，我们假设外部线索 涉及配体浓度和与细胞外基质成分的相互作用， 受体迅速脱敏的情况和它保持响应的情况，继续迁移， 并有效清除趋化因子。本建议的目的是实现一个全面的和预测性的 基于系统的理解调节CCR 2迁移的信号传导和贩运机制， 清除功能。在这里，我们提出了三个目标：（i）我们将描绘CCR 2的关键换能器的作用， 通过结合药理学抑制和基因沉默与细胞介导的迁移和清除 基于迁移和清除的测定以及受体运输的荧光成像。我们还将进一步 定义调节受体从激活后脱敏到维持 反应和成为有效的清道夫;（ii）我们将发现和表征新的蛋白质， 使用基于无偏质谱法的方法调节CCR 2迁移和清除， 正交分析方法和布尔网络建模，以确定验证的关键调节器的优先顺序，如 目标1;（iii）我们将开发基于计算机网络的CCR 2信号传导和贩运预测模型 使用布尔反应-应急网络，最初由文献提供信息，并从系统数据中进行改进 在目标1和2中收集。该提案的预期结果是预测性的和时空解决的 CCR 2的相互作用组和信号网络，揭示了目前未知的功能和调节 单核细胞迁移和清除机制。

Monocyte migration into tissues, guided by inflammatory chemokine CCL2 and its G protein-coupled receptor CCR2, is key to both normal physiological responses and many diseases including neurodegenerative disease, traumatic brain injury, arthritis, diabetic nephropathy and non-alcoholic fatty liver disease. Moreover, CCR2 is currently the target of ongoing clinical trials for diabetic nephropathy and pancreatic cancer. Given the therapeutic significance of CCR2, it is surprising that its role in monocyte function is so poorly understood. In particular, while CCR2 is well-recognized to promote cell movement, what is largely unappreciated, is that it has a second, equally important function, whereby it scavenges chemokines from the extracellular medium by internalizing and trafficking chemokine to lysosomes for degradation. Scavenging may enable receptor- expressing cells to move along gradients of increasing chemokine concentration without desensitizing; it may also modulate the responsiveness of receptors on different cells by altering the availability of specific chemokines, thereby regulating whether and what type of cells migrate. We hypothesize that it is, in fact, the cohesive integration of these two functions (cell movement and scavenging) that enables CCR2 to effectively control directional cell migration in different biological contexts. Moreover, we hypothesize that external cues involving ligand concentration and interactions with extracellular matrix components define a switch between a scenario where the receptor rapidly desensitizes and one where it remains responsive, continues to migrate, and efficiently scavenges chemokine. The objective of this proposal is to achieve a comprehensive and predictive systems-based understanding of the signaling and trafficking mechanisms that regulate CCR2 migration and scavenging functions. Here we propose three Aims: (i) We will delineate the roles of key transducers of CCR2- mediated migration and scavenging by combining pharmacological inhibition and gene silencing with cell based assays of migration and scavenging and fluorescence imaging of receptor trafficking. We will also further define the mechanisms that regulate the switch from receptors being desensitized upon activation to maintaining responsiveness and becoming efficient scavengers; (ii) We will discover and characterize novel proteins that regulate CCR2 migration and scavenging using unbiased Mass Spectrometry based approaches coupled with orthogonal methods of analysis and Boolean network modeling to prioritize key regulators for validation as in Aim 1; (iii) We will develop predictive in silico network-based models of CCR2 signaling and trafficking using Boolean reaction-contingency networks initially informed by literature with refinement from systematic data collected in Aims 1 and 2. The expected outcome of this proposal is predictive and spatiotemporally-resolved interactome and signaling network of CCR2 that reveals currently unknown functional and regulatory mechanisms of monocyte migration and scavenging.