Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding.
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.
Wu
L
L
LeukoSite, Inc., Cambridge, Massachusetts 02142, USA. lijun_wu@leukosite.com
LaRosa
G
G
Kassam
N
N
Gordon
C J
CJ
Heath
H
H
Ruffing
N
N
Chen
H
H
Humblias
J
J
Samson
M
M
Parmentier
M
M
Moore
J P
JP
Mackay
C R
CR
D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D000918:Antibody Specificity; D001667:Binding, Competitive / Q000276:immunology; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000276:immunology / Q000502:physiology; D019742:Chemokines, CC / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008024:Ligands; D016399:Lymphoma, T-Cell; D019402:Macrophage Inflammatory Proteins / Q000276:immunology / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D019742:Chemokines, CC; D015699:HIV Envelope Protein gp120; D008024:Ligands; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5