Temporal Proteomics Profiling of Lipid Rafts in CCR6-Activated T Cells Reveals the Integration of Actin Cytoskeleton Dynamics

Abstract

Chemokines orchestrate leukocyte migration toward sites of inflammation and infection and target leukocytes via chemokine receptors such as CCR6, a subfamily of the seven-transmembrane G-protein-coupled receptors. Lipid rafts are cholesterol and sphingolipid-enriched liquid-ordered membrane microdomains thought to serve as scaffolding platforms for signal transduction. To globally understand the dynamic changes of proteins within lipid rafts upon CCR6 activation in T cells, we quantitatively analyzed the time-dependent changes of lipid raft proteome using our recently reported membrane proteomics strategy combining gel-assisted digestion, iTRAQ labeling and LC-MS/MS. To our knowledge, the error-free identification of 852 proteins represents the first data set of the raft proteome in T cells upon chemokine receptor activation, including 354 previously annotated raft proteins and 85 dynamically recruited proteins that are potential raft-associated proteins. The temporal profiles revealed that many proteins involved in the actin cytoskeleton rearrangement are actively recruited into lipid rafts upon CCR6 activation. We further confirmed the proteomics results by Western blotting and used small interfering RNA-mediated knockdown to evaluate their roles upon CCR6 activation. In sum, we employed quantitative proteomic strategy to analyze raft proteome and identified many molecules actively involved in the control of actin assembly and disassembly regulating CCR6 activation-induced cell migration.

CCL20 and β-Defensin-2 Induce Arrest of Human Th17 Cells on Inflamed Endothelium In Vitro under Flow Conditions

“…Another possible mechanism underlying the modulation of the expression of this chemokine receptor might involve lipid rafts, membrane-bound microdomains that are enriched with cholesterol, sphingolipids, and saturated fatty acids and that function as a scaffolding platform for a variety of cellular functions, including signal transduction events (57). It has recently been shown that cholesterol depletion of CCR6-expressing Jurkat T cells impairs CCL20-mediated CCR6 signaling, suggesting that lipid rafts are necessary for CCR6 activation (58). It can therefore be postulated that Ag-specific activation of CCR6-expressing T cells might result in a remodeling of the distribution of CCR6 at the cell surface, a subsequent exclusion of the receptor from the rafts and transport to clathrin-coated pits, thereby facilitating its internalization in the endosome and its subsequent degradation by the proteasome.…”

A Proteomic Approach to Screening of Dynamic Changes in Detergent-Resistant Membranes from Activated Human Primary T Cells

“…However, quantitative changes in protein levels are not covered by this approach and are also of functional importance in signal transduction. Quantitative proteomic approaches dissecting antigen receptor-mediated dynamics of DRMs have been reported [11,35,36]. However, these studies were performed on material obtained from cell lines and have been based mostly on metabolic labeling of cells by differential feeding with light or heavy isotopic amino acids, an approach which is not possible with primary cells.…”

Implementation of a Flow Cytometry Strategy to Isolate and Assess Heterogeneous Membrane Raft Domains

“…Proteomic raft analysis can be achieved by extracting these domains from the plasma membrane (Foster et al, 2003, Jordan & Rodgers, 2003, Lin et al, 2010, Mannova et al, 2006, Nebl et al, 2002. Isolation of membrane rafts was traditionally performed in cold, non-ionic detergent, such as Triton X-100, in which raft proteins are largely insoluble (Brown & Rose, 1992).…”