Lipids in Maintenance of Chemokine and T-Cell Receptor

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Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors that are differentially expressed by a number of immune and non-immune cell populations. Certain CRs have been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. Lipid rafts are believed to be important signaling platforms enriched in many signaling proteins, including but not limited to src kinases, Ga subunit, H-Ras, LAT, and NOS. CCR5 and CXCR4 have been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 and CXCR4 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of SDF-1 and MIP-1b using CXCR4- or CCR5-expressing T cells, respectively. Oxidized forms of cholesterol, known as oxysterols, are abundant in various food products and can be found naturally in membranes and mitochondria of a variety of cell types. Cholesterol oxidation was also found to result in the loss of chemokine binding and function in T cells and monocytes. Reloading treated cells with non-oxidized cholesterol restores chemokine binding and function in these situations. Antibodies specific for distinct CXCR4 or CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction and cholesterol oxidation. Moreover, bindings studies with labeled chemokines have demonstrated extensive co-localization of ligand binding with the GM1 lipid raft marker while using anti-chemokine receptor antibodies, we found the majority of chemokine receptors co-localize with CD59 and only partially with GM1. These results suggest that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of chemokine receptors. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the chemokine receptor conformation and are necessary for both the binding and function of this chemokine receptor. This cholesterol and lipid raft requirement for ligand binding may play a significant physiological role in controlling immune cell signaling and migration. More specific efforts are also underway examining the differences in the make-up of lipid rafts within the cell membranes of young and aged lymphocytes. Given the large number of alterations in lipid and peroxidation and metabolism with age, changes in the types, saturation and levels of various membrane sphingolipids, fatty acids and cholesterol may result in specific changes in membrane fluidity, protein association and aggregation, cellular activation and function. In addition, through HPLC separation, 2-D gel electrophoresis and mass spectroscopy, additional studies are underway creating a proteomic expression profile of the various proteins within the lipid rafts of young and aged lymphocytes at various stages of activation. We believe that a greater understanding of the various signaling and cell surface proteins associated with lipid rafts may provide great insight into age-related alterations in cell signaling and migration.