Abstract 15386: Integrated Phosphoproteome and Transcriptome Analysis of Biased Agonists at CXCR3 Reveals Differential Cellular Outputs of Inflammation

Abstract

Background and Objective: G-protein-coupled receptors (GPCRs) are the most common cell surface receptor class and are known to influence nearly every biological process. GPCRs signal through downstream effectors such as G proteins and β-arrestins. Certain ligands drive activation of G protein over β-arrestin signaling, or vice versa - a phenomenon known as biased agonism. To investigate the mechanisms and implications of biased agonism, we studied CXCR3, a chemokine receptor expressed on T cells, which binds three biased endogenous ligands, CXCL9, CXCL10, and CXCL11. Importantly, CXCR3 has been implicated in atherosclerosis and other inflammatory diseases. Methods: To obtain a broad view of biased signaling at CXCR3, we performed global phosphoproteomic and transcriptomic analyses of CXCR3-transfected HEK 293 cells and activated human CD8+ T-cells, respectively, treated with CXCL9, CXCL10, and CXCL11. To assess the phosphoproteome, we utilized an antibody-free mass spectrometry approach with high-pressure, high-resolution separation and intelligent selection. RNA-seq was performed to assess transcriptomic changes in activated CD8+ T-cells. Results: Our analyses revealed 1532 phosphosites and 887 transcripts differentially regulated between treatment groups. Gene Set Enrichment Analysis (GSEA) of CXCL11-treated cells revealed enrichment of pro-inflammatory targets including the Myc and TNF-α pathways via NFKB not observed in CXCL10- or CXCL9-treated cells. GSEA of CXCL10- and CXCL11-treated cells also revealed enrichment of the IL6-JAK-STAT3 pathway, linked to chronic inflammatory conditions. Kinase-Substrate Enrichment Analysis (KSEA) revealed differential phosphorylation activity associated with multiple kinases including AURKB (CXCL9), ROCK1 (CXCL9, 10, 11), CDK9 (CXCL10), and ATM (CXCL10, 11) - all known to regulate pro-inflammatory targets. Several phosphoproteins were transcription factors, including p53, known to mediate IL-6 production and atherosclerosis pathogenesis. Conclusion: Together, our integrated analysis of phosproteomic and transcriptomic data reveal the extent of differential cellular outputs produced by biased agonists at CXCR3 in modulating targets implicated in inflammatory diseases.