Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding

Abstract

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)-1alpha, and MIP-1beta, 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-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha 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.

Figure 2

Reactivity of CCR5-specific mAbs with CCR5/CCR2b receptor chimeras. The structures of CCR5/CCR2b chimeras used in this study are shown schematically on the left-hand side. Regions derived from CCR5 are shown in light gray, and regions derived from CCR2b are shown in black. Stable Chinese hamster ovary cell transfectants expressing various CCR5/CCR2b receptor chimeras were stained with anti-CCR5 mAb 3A9, 5C7, 2D7, anti-CCR2b mAb 5A11, or anti-CXCR1 mAb 7D9. The level of staining of the transfectants by the various mAbs was graded + to +++, or negative (neg).

Figure 1

mAb 2D7 recognizes CCR5 specifically. (A) Reactivity of mAb 2D7 with CCR5 L1.2 cells, but not with CXCR4 L1.2 cells. (B) Two color staining of human PBL with 2D7 (green fluorescence, x-axis) and 12G5 (anti-CXCR4, red fluorescence, y-axis). Quadrants were set on the basis of control and single color stainings.

Figure 3

mAb 2D7 inhibits the binding of ¹²⁵I–MIP-1α, –MIP-1β, and -RANTES to CCR5. CCR5 L1.2 cells (A), THP1 cells (B), or day 15–activated IL-2–stimulated CD3 blast T cells (C) were incubated with 0.1 nM ¹²⁵I-labeled–MIP-1α, –MIP-1β, or -RANTES, in the absence (total binding) or presence of either 10 μg/ml of mAb 2D7 (an IgG1 isotype), mAb 3A9, control IgG1 mAb, or 100 nM unlabeled chemokine. After 45–60 min at room temperature, cells were washed and counted as described in Materials and Methods. Data are shown as the percentage of total binding, i.e., in the absence of mAb or unlabeled chemokines.

Figure 4

mAb 2D7 inhibits [Ca²⁺]_i flux in CCR5 L1.2 cells in response to MIP-1α. CCR5 L1.2 cells were labeled with Fura-2 as described in Materials and Methods, and stimulated sequentially with mAb, followed 40 s later with MIP-1α, and 100 s with SDF-1. [Ca²⁺]_i fluorescence changes were recorded using a spectrofluorometer. The tracings were representative of three separate experiments. In the top panel, an irrelevant mAb (MOPC-21) was used, and in the bottom panel, mAb 2D7. Antibodies were used at a final concentration of 20 μg/ml. MIP-1α was used at 100 nM and SDF-1 was used at 200 nM.

Figure 5

Inhibition of chemotactic responses of various cell types to MIP-1α, MIP-1β, and RANTES, using mAb 2D7. A, CCR5 L1.2 cells; B, blood lymphocytes; C, blood monocytes; and D, day 21 activated, IL-2–stimulated T cells. For these experiments, 10⁶ cells were placed in the top chamber of the Transwell and an optimal concentration of chemokine (usually 12.5 nM) was placed in the bottom chamber. Various concentrations of 2D7 mAb were placed in the top well. After various periods of time (1–4 h) the cells migrating to the bottom chamber were counted using flow cytometry. The results are representative of at least four separate experiments. Untransfected L1.2 cells showed no migration to MIP-1α, MIP-1β, or RANTES (data not shown). Chemotactic index was calculated by dividing the number of migrated cells in response to a specific chemokine by that in the absence of chemokine (background). The background values for these cells are: 28 (CCR5 L1.2), 136 (lymphocytes), 507 (monocytes), and 615 (CD3 blasts).

Figure 6

Inhibition of ¹²⁵I-gp120 binding and HIV-1 infection by anti-CCR5 mAbs. (A) Inhibition of ¹²⁵I-labeled M-tropic HIV-1 JR-FL gp120 binding to CCR5 L1.2 transfectants by mAb 2D7 and 3A9. CCR5 L1.2 cells were incubated with 0.2 nM ¹²⁵I-labeled gp120 and 20 nM sCD4 in the absence or presence of increasing concentrations of mAb 2D7 or 3A9. After 60 min at room temperature, cells were washed and counted as described in Materials and Methods. An IgG1 control mAb was used as a control. 100% of inhibition was defined as that caused by 100 nM of unlabeled gp120. (B) Inhibition of HIV-1 infection in U87-CD4-CCR5 cells by mAb 2D7. The infectability of U87-CD4-CCR5 cells by M-tropic (ADA and JR-FL), dual-tropic (DH123), and T-tropic (HxB2) HIV-1 strains, in the absence or presence of increasing concentrations of 2D7 or 50 μg/ml of an IgG1 control mAb, was determined using a virus entry assay based on single-cycle infection as described in Materials and Methods. Infection of the cells was measured by quantification of luciferase activity.