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 either CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors (GPCRs) that are differentially expressed by a number of immune and non-immune cell populations. CCR5 has 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. Signal transduction through the T and B cell receptors as well as the IgE receptor involves the recruitment of signaling assemblies to lipid rafts. CCR5 has been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. This receptor has also recently been shown to be palmitoylated which is one of the important modifications in lipid raft targeting of proteins. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of MIP-1b using CCR5-expressing T cells. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored MIP-1b binding to BCD-treated cells. Antibodies specific for distinct CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction. Moreover, cells stained with fluorescently-labeled MIP-1b extensively co-localized with the GM1 lipid raft marker while using anti-CCR5 antibodies, the majority of CCR5 on these cells co-localized with CD59 and only partially with GM1 suggesting that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of CCR5. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the CCR5 conformation and are necessary for both the binding and function of this chemokine receptor. Similar studies were performed using examining the role of cholesterol in CXCR4 function. We have determined that cholesterol extraction by beta-cyclodextrin (BCD) also inhibits the CXCR4 ligand, SDF-1a, binding to CXCR4 on human T cells. Intracellular calcium responses to SDF-1a, as well as receptor internalization, were similarly impaired in treated T cells. Loss in ligand binding appears to be due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1a binding was effectively restored by reloading cholesterol. These data, along with microscopic evidence that SDF-1a binds within lipid rafts, suggests that cholesterol is essential for CXCR4 function and conformational integrity within lipid rafts.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000767-01
Application #
6531203
Study Section
(LI)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Jun, Do Youn; Taub, Dennis; Chrest, Francis J et al. (2014) Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation. Cell Immunol 287:78-85
Ghosh, Manik C; Collins, Gary D; Vandanmagsar, Bolormaa et al. (2009) Activation of Wnt5A signaling is required for CXC chemokine ligand 12-mediated T-cell migration. Blood 114:1366-73
Ghosh, Manik C; Baatar, Dolgor; Collins, Gary et al. (2009) Dexamethasone augments CXCR4-mediated signaling in resting human T cells via the activation of the Src kinase Lck. Blood 113:575-84
Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok et al. (2008) 17Alpha-estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells. Toxicol Appl Pharmacol 231:401-12
Giri, Banabihari; Dixit, Vishwa D; Ghosh, Manik C et al. (2007) CXCL12-induced partitioning of flotillin-1 with lipid rafts plays a role in CXCR4 function. Eur J Immunol 37:2104-16
Nguyen, Dzung H; Giri, Banabihari; Collins, Gary et al. (2005) Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement. Exp Cell Res 304:559-69
Nguyen, Dzung H; Espinoza, Juan C; Taub, Dennis D (2004) Cellular cholesterol enrichment impairs T cell activation and chemotaxis. Mech Ageing Dev 125:641-50
Nguyen, Dzung H; Taub, Dennis D (2003) Membrane incorporation of 22-hydroxycholesterol inhibits chemokine receptor activity. Exp Cell Res 285:268-77
Nguyen, Dzung H; Taub, Dennis D (2003) Inhibition of chemokine receptor function by membrane cholesterol oxidation. Exp Cell Res 291:36-45
Nguyen, Dzung H; Taub, Dennis (2002) Cholesterol is essential for macrophage inflammatory protein 1 beta binding and conformational integrity of CC chemokine receptor 5. Blood 99:4298-306

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