Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Characterisation of chemokine GPCRs signalling in monocytes using state-of-the-art phosphoproteomics approach (#169)

Rina Pokhrel 1 2 , Simon Foster 1 2 , Anup Shah 2 3 , Ralf Schittenhelm 2 3 , Martin Stone 1 2
  1. Biomedicine Discovery Institute & Department of Molecular Biology and Biochemistry, Monash University, Clayton, VIC, Australia
  2. Monash University, Clayton, VIC, Australia
  3. Monash Biomedical Proteomics Facility and Monash Bioinformatics Platform, Monash University, Clayton, VIC, Australia

Introduction. Chemokine receptors are G protein-coupled receptors (GPCRs) expressed on the surface of leukocytes that bind to chemoattractant cytokines called chemokines. They are the key player in inflammation. Chemokine-receptors interaction is a crucial step in recruiting monocytes at the inflammation site for regulation of inflammatory responses. Monocytes abundantly express CCR1 and CCR2 receptors, which are potential therapeutic targets in inflammatory diseases including atherosclerosis and rheumatoid arthritis. Lack of effective anti-inflammatory drugs and failure of many clinical trials till date could be due to complexity in the chemokine-receptor signalling network.

Aim. To characterise the signalling network of robustly activated chemokine receptors in monocytes using state-of-the-art phosphoproteomics and computational modelling approaches. 

Methods. We carried out well-established cell-signalling assays, including chemotaxis (96- well MultiScreen plates, Merck) and ERK phosphorylation (AlphaLISA Surefire Ultra, Perkin Elmer) in monocyte-like THP-1 cells expressing CCR1. To characterise downstream CCR1-dependent signalling events, we performed a phosphoproteomics study using data-dependent acquisition (DDA) mass spectrometry to quantify changes in phosphopeptides between untreated and CCL5-stimulated THP-1 cells. 

Results. We have previously demonstrated that the chemokines, CCL5 and CCL7 elicited a concentration-dependent increase in chemotaxis of THP-1 cells and phosphorylation of ERK. A CCL5 stimulation time course monitored by phosphoproteomics, revealed regulation of 931 phosphosites and 534 unique proteins (1-way ANOVA with FDR ˂0.05), including phosphorylation of CXCR2. A similar study showed that treatment with CCL2, acting via the receptor CCR2, resulted in regulation of 460 phosphosites and 329 unique proteins, including phosphorylation of CCR1.

Discussion. These findings provide critical insights into complexity of chemokine receptor signalling cascade. The extensive signalling network identified from protein phosphorylation data includes expected chemokine receptor signalling pathways as well as previously unrecognised signalling mechanisms therefore, result to potential novel target related to CCR1 and CCR2 signalling.