CCR5 Conformation Research Articles

Inhibition of chemokine receptor function by membrane cholesterol oxidation

Membrane cholesterol is required to maintain chemokine receptor conformation and function for CXCR4 and CCR5. We previously demonstrated that chemokines preferentially bind to receptors within lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains. To further elucidate the role of cholesterol in chemokine receptor function, we examined the effects of membrane cholesterol oxidation by cholesterol oxidase (CO), which enzymatically converts cholesterol to 4-cholesten-3-one. Here, we demonstrate that CO treatment (0.25–2.0 U/ml) of human T cells inhibits CXCL12 (SDF-1α) and CCL4 (MIP-1β) binding to cell surface CXCR4 and CCR5, respectively, resulting in the inhibition of chemokine-mediated intracellular calcium mobilization and chemotaxis. The effects were significantly enhanced by cotreatment with low-dose sphingomyelinase (SMase) (0.125 mU/ml), which produced little inhibitory effect by itself. CO and SMase treatment also inhibited HIV-1 infection through CXCR4, but not virus replication. Similar to the removal of membrane cholesterol, CO/SMase treatment induced conformation changes in the chemokine receptors as detected by differential loss in binding of epitope-specific monoclonal antibodies. We conclude that the native form of cholesterol with the hydroxyl group at C3 is critical to CXCR4 and CCR5 conformation and function.

Cholesterol is essential for macrophage inflammatory protein 1β binding and conformational integrity of CC chemokine receptor 5

The chemokine receptor, CCR5, is used as a human immunodeficiency virus coreceptor in combination with CD4 during transmission and early infection. CCR5 has been shown to be palmitoylated and targeted to cholesterol- and sphingolipid-rich membrane microdomains termed "lipid rafts." However, the role of cholesterol and lipid rafts on chemokine binding and signaling through CCR5 remains unknown. We found that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) significantly reduced the binding and signaling of macrophage inflammatory protein 1 beta (MIP-1 beta) using CCR5-expressing CEM-NKR T cells. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored MIP-1 beta binding to BCD-treated cells. Antibodies specific for distinct CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction to varying degrees. Moreover, cells stained with fluorescently labeled MIP-1 beta extensively colocalized with the GM1 lipid raft marker while using anti-CCR5 antibodies; most of CCR5 on these cells only partially colocalized 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.

Determinants of the trans-Dominant Negative Effect of Truncated Forms of the CCR5 Chemokine Receptor

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in the CCR5 gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Delta32) lacking the last three transmembrane domains (TM5-7). This mutant is retained in the endoplasmic reticulum and exerts a trans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Delta32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Delta32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Delta32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Delta32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Delta32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.

Extracellular Cysteines of CCR5 Are Required for Chemokine Binding, but Dispensable for HIV-1 Coreceptor Activity

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.