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

Wu L, LaRosa G, Kassam N, Gordon CJ, Heath H, Ruffing N, Chen H, Humblias J, Samson M, Parmentier M, Moore JP, Mackay CR - J. Exp. Med. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: LeukoSite, Inc., Cambridge, Massachusetts 02142, USA. lijun_wu@leukosite.com

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.

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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).
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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).

Mentions: We sought to generate mAbs to CCR5 to inhibit the various functions of this molecule, and to understand how different CCR5 domains bind chemokines and HIV-1. We have previously described anti-CCR5 mAbs that inhibit HIV-1 binding, but not chemokine binding (19). New mAbs to CCR5 were generated by immunizing C57BL6 mice with the murine pre–B cell lymphoma line, L1.2, which expresses high levels of transfected human CCR5. An mAb, termed 2D7, reacted with CCR5-transfected L1.2 cells, as well as Chinese hamster ovary cells expressing certain portions of CCR5 (see Fig. 2), but not with L1.2 cells expressing CXCR4 (Fig. 1) or various other receptors, including CCR2b (not shown). Moreover, 2D7 showed an identical pattern of reactivity against human leukocytes, as previously noted for our other anti-CCR5 mAbs (19, 48). In particular, it stained mostly the CXCR4− subset of human PBL (Fig. 1 B), as well as a subset of tissue macrophages (not shown).


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

Wu L, LaRosa G, Kassam N, Gordon CJ, Heath H, Ruffing N, Chen H, Humblias J, Samson M, Parmentier M, Moore JP, Mackay CR - J. Exp. Med. (1997)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199098&req=5

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).
Mentions: We sought to generate mAbs to CCR5 to inhibit the various functions of this molecule, and to understand how different CCR5 domains bind chemokines and HIV-1. We have previously described anti-CCR5 mAbs that inhibit HIV-1 binding, but not chemokine binding (19). New mAbs to CCR5 were generated by immunizing C57BL6 mice with the murine pre–B cell lymphoma line, L1.2, which expresses high levels of transfected human CCR5. An mAb, termed 2D7, reacted with CCR5-transfected L1.2 cells, as well as Chinese hamster ovary cells expressing certain portions of CCR5 (see Fig. 2), but not with L1.2 cells expressing CXCR4 (Fig. 1) or various other receptors, including CCR2b (not shown). Moreover, 2D7 showed an identical pattern of reactivity against human leukocytes, as previously noted for our other anti-CCR5 mAbs (19, 48). In particular, it stained mostly the CXCR4− subset of human PBL (Fig. 1 B), as well as a subset of tissue macrophages (not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: LeukoSite, Inc., Cambridge, Massachusetts 02142, USA. lijun_wu@leukosite.com

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.

Show MeSH
Related in: MedlinePlus