Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

中文翻译：

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趋化因子受体CCR2激活导致信号网络的磷酸化蛋白质组学表征。

白细胞募集是组织炎症的普遍特征，并受趋化因子与其G蛋白偶联受体相互作用的调节。CC趋化因子受体2（CCR2）的相关同源趋化因子配体（包括CC趋化因子配体2（CCL2））的激活在几种炎症性疾病的单核细胞募集中起着核心作用。在这项研究中，我们使用磷酸化蛋白质组学对CCR2激活CCR2所产生的信号网络进行了无偏性表征。使用独立于数据的采集MS分析，我们量化了Clp2激活后六个时间点稳定表达CCR2的FlpIn-HEK293T细胞中的蛋白质组和磷酸化蛋白质组。差异表达分析确定了441个蛋白上的699个显着调节的磷酸化位点。不出所料 已知这些蛋白中的许多蛋白都参与规范的信号转导途径和肌动蛋白细胞骨架动力学的调节，包括许多鸟嘌呤核苷酸交换因子和GTPase激活蛋白。此外，我们确定了在细胞核中起作用的许多蛋白质中受调节的磷酸化位点，包括核孔复合体的几种成分。这项研究的结果为CCR2信号传递提供了前所未有的详细水平，并确定了调节CCR2功能的潜在靶标。包括核孔复合体的几种成分。这项研究的结果为CCR2信号传递提供了前所未有的详细水平，并确定了调节CCR2功能的潜在靶标。包括核孔复合体的几种成分。这项研究的结果为CCR2信号传递提供了前所未有的详细水平，并确定了调节CCR2功能的潜在靶标。