Inflammation is the body’s response to injury or infection. A hallmark of inflammation is the accumulation of leukocytes, which remove pathogens and necrotic tissue by phagocytosis and proteolytic degradation. Leukocytes are mainly recruited by chemokine activation of chemokine receptors, resulting in leukocyte morphological changes, extravasation into the inflamed tissue and chemotaxis along the chemokine gradient to the site of injury or infection. However, dysregulation of leukocytes/monocytes can result in inflammatory diseases. Therefore, chemokine receptors and chemokines are potential therapeutic targets in a wide range of inflammatory diseases.
CCL2 (or monocyte chemoattractant protein-1, MCP-1) and CCR2 is the major chemokine and receptor pair, involved in recruitment of monocytes, which subsequently differentiate into macrophages, contributing to the pathogenesis of atherosclerosis, obesity and type 2 diabetes. Considering the importance of CCL2-CCR2 in these diseases, there is strong motivation to understand their mechanism of activation and signalling. In particular, while CCR2 is known to signal via G protein and β-arrestin-mediated pathways, the downstream signalling pathways have not been thoroughly explored.
Protein phosphorylation and dephosphorylation are crucial for cellular signal transduction. Dynamic regulation of reversible, site-specific protein phosphorylation is critical to the signalling networks. Here, we exploited phosphoproteomic analyses in combination with cutting-edge data-independent acquisition (DIA)-mass spectrometry to unravel signal transduction events and their dynamic regulation in CCL2-activated CCR2-expressing cells. We created a library containing more than 25,000 phosphopeptides. During the first hour of CCL2-activation, we reproducibly and accurately quantified ~15,000 phosphopeptides in each repeat without any fractionation. From this study, we identified the key components and major pathways involved in CCR2 signalling.
Given the importance of CCL2-CCR2 signalling in the pathogenesis of many debilitating and prevalent diseases, this study does not only provide in-depth and novel insights into this signalling pathway, but will also guide the identification of potential therapeutic targets.