Wu L, LaRosa G, Kassam N, Gordon C J, Heath H, Ruffing N, Chen H, Humblias J, Samson M, Parmentier M, Moore J P, Mackay C R
LeukoSite, Inc., Cambridge, Massachusetts 02142, USA.
J Exp Med. 1997 Oct 20;186(8):1373-81. doi: 10.1084/jem.186.8.1373.
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.
CCR5是一种由T细胞和巨噬细胞表达的趋化因子受体，它也是HIV-1巨噬细胞（M）嗜性毒株的主要共受体。为了了解趋化因子和HIV-1与CCR5结合的分子基础，我们制备了多种抑制CCR5各种相互作用的单克隆抗体，并使用一组CCR5/CCR2b嵌合体绘制了这些单克隆抗体的结合位点。一种名为2D7的单克隆抗体完全阻断了CCR5的三种天然趋化因子配体，即调节激活正常T细胞表达和分泌因子（RANTES）、巨噬细胞炎性蛋白（MIP）-1α和MIP-1β与CCR5转染细胞的结合及趋化作用。该单克隆抗体是CCR5的真正拮抗剂，因为它未能刺激CCR5转染细胞内钙浓度升高，但能阻断RANTES、MIP-1α或MIP-1β引发的钙反应。该单克隆抗体抑制了活化T细胞的大部分RANTES和MIP-1α趋化反应，但未抑制单核细胞的趋化反应，这表明这两种细胞类型对趋化因子受体的使用存在差异。2D7的结合位点定位于CCR5的第二个细胞外环，而一组未能阻断趋化因子结合的单克隆抗体均定位于CCR5的NH2末端区域。识别第二个细胞外环或NH2末端区域的单克隆抗体均可有效抑制M嗜性HIV-1衍生包膜糖蛋白gp120与CCR5的结合，尽管前者显示出更强的抑制作用。此外，2D7在体外有效阻断了几种M嗜性和双嗜性HIV-1毒株的感染性。这些结果表明HIV-1 gp120与CCR5不同区域的结合模式复杂，但趋化因子结合模式相对简单。我们得出结论，CCR5的第二个细胞外环是开发趋化因子或HIV-1与CCR5结合抑制剂的理想靶点。
