CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α, and MIP-1β, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1α, or MIP-1β. This mAb inhibited most of the RANTES and MIP-1α chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1–derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.

The CC-chemokine RANTES (regulated on activation normal T-cell expressed and secreted; CCL5) transduces multiple intracellular signals. Like all chemokines, it stimulates G protein-coupled receptor (GPCR) activity through interaction with its cognate chemokine receptor(s), but in addition also activates a GPCR-independent signaling pathway. Here, we show that the latter pathway is mediated by an interaction between RANTES and glycosaminoglycan chains of CD44. We provide evidence that this association, at both low, physiologically relevant, and higher, probably supraphysiologic concentrations of RANTES, induces the formation of a signaling complex composed of CD44, src kinases, and adapter molecules. This triggers the activation of the p44/42 mitogen-activated protein kinase (MAPK) pathway. By specifically reducing CD44 expression using RNA interference we were able to demonstrate that the p44/p42 MAPK activation by RANTES requires a high level of CD44 expression. As well as potently inhibiting the entry of CCR5 using HIV-1 strains, RANTES can enhance HIV-1 infectivity under certain experimental conditions. This enhancement process depends in part on the activation of p44/p42 MAPK. Here we show that silencing of CD44 in HeLa-CD4 cells prevents the activation of p44/p42 MAPK and leads to a substantial reduction in HIV IntroductionThe chemokine RANTES (regulated on activation normal T-cell expressed and secreted; CCL5) is a member of the CC-chemokine family, a group of small proteins with a highly conserved tertiary. 1,2 Chemokines recruit and activate specific leukocyte populations. Multiple chemokine receptors with partially overlapping specificities for ligand binding have been described, suggesting a possible redundancy of the system. However, an increased selectivity of the action of chemokines is thought to be gained through their ability to discern and preferentially bind to certain glycosaminoglycan (GAG) subpopulations, a process considered important in mediating tissue-specific leukocyte recruitment. [3][4][5][6] The chemokine RANTES has a complex influence on the biology of a variety of cell types including T lymphocytes, monocytes, natural killer cells, dendritic cells, basophils, and eosinophils. 7,8 At nanomolar concentrations, RANTES binds to and activates several 7-transmembrane G protein-coupled receptors (GPCRs), namely the chemokine receptors CCR1, CCR3, and CCR5. Ligation of these receptors activates a heterotrimeric G␣i protein-coupled signaling pathway, characterized by a transient Ca 2ϩ influx, 7,9 and triggers activation of cell polarization and chemotaxis. 10 In addition to these classic chemokine-activated responses, RANTES also induces several biochemical and biologic effects that are to date unique to this chemokine and that are triggered through a GPCR-independent pathway. Induction of this pathway is mediated by protein tyrosine kinases (PTKs), occurs at high micromolar concentrations of the chemokine, and leads to a sustained influx of Ca 2ϩ . 9 We and others have previously demonstr...