Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.Entry of human immunodeficiency virus-1 (HIV-1) 2 into cells requires the formation of entry complexes involving the viral envelope glycoprotein gp120 and the target cell receptors CD4 and either CCR5 or CXCR4. CCR5 and CXCR4 are G-protein-coupled chemokine receptors that play critical roles in immune responses (1-4). Considerable effort has been focused on the development of therapeutics targeting chemokine receptors to block HIV entry (5, 6). A major problem in developing safe and effective co-receptor inhibitors is the risk of interfering with chemokine receptor signaling, thereby causing harmful side effects. Understanding the molecular mechanisms underlying chemokine function in HIV infection will hopefully provide a foundation for designing and screening drugs that offer strong and long lasting HIV-inhibitory function but have little effect on the physiology of the homeostatic chemokine system. In the past years, extraordinary progress has been made in solving the structures of gp120 and CD4 and in demonstrating that chemokines and small derivative molecules block HIV infection (4, 6 -11). Yet, the details regarding gp120-induced formation of HIV entry complexes in the context of live cells still need to be resolved.The plasma membrane of eukaryotic cells consists of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments known as lipid or non-lipid raft microdomains (12-17). Lipids in rafts possess long and saturated fatty acyl chains and are organized in a tightly packed, liquid-ordered manner, whereas non-lipid raft microdomains contain shorter, unsaturated fatty acyl chains and are in a loosely packed, disordered state (12-17). Lipid rafts are defined as microdomains that are enriched with cholesterol, glycosphingolipids, and sphingomyelin and are often isolated in detergent-resistant membrane fractions. Previous studies draw different conc...

BackgroundDimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation.Methodology/Principal FindingsUsing a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5STA and CCR5Δ4.Conclusions/SignificanceTaken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.

Human leukocytes, including macrophages and neutrophils, are phagocytic immune cells that capture and engulf pathogens and subsequently destroy them in intracellular vesicles. To accomplish this vital task, these leukocytes utilize two basic cell behaviors-chemotaxis for chasing down infectious pathogens and phagocytosis for destroying them. The molecular mechanisms controlling these behaviors are not well understood for immune cells. Interestingly, a soil amoeba, Dictyostelium discoideum, uses these same behaviors to pursue and injest its bacterial food source and to organize its multi-cellular development. Consequently, studies of this model system have provided and will continue to provide us with mechanistic insights into the chemotaxis and phagocytosis of immune cells. Here, we review recent research in these areas that have been conducted in the Chemotaxis Signal Section of NIAID's Laboratory of Immunogenetics.