Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies.
CC-chemokine receptor 5 (CCR5) is the principal coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have generated a set of anti-CCR5 monoclonal antibodies and characterized them in terms of epitope recognition, competition with chemokine binding, receptor activation and trafficking, and coreceptor activity. MC-4, MC-5, and MC-7 mapped to the amino-terminal domain, MC-1 to the second extracellular loop, and MC-6 to a conformational epitope covering multiple extracellular domains. MC-1 and MC-6 inhibited regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory polypeptide-1beta, and Env binding, whereas MC-5 inhibited macrophage inflammatory polypeptide-1beta and Env but not RANTES binding. MC-6 induced signaling in different functional assays, suggesting that this monoclonal antibody stabilizes an active conformation of CCR5. Flow cytometry and real-time confocal microscopy showed that MC-1 promoted strong CCR5 endocytosis. MC-1 but not its monovalent isoforms induced an increase in the transfer of energy between CCR5 molecules. Also, its monovalent isoforms bound efficiently, but did not internalize the receptor. In contrast, MC-4 did not prevent RANTES binding or subsequent signaling, but inhibited its ability to promote CCR5 internalization. These results suggest the existence of multiple active conformations of CCR5 and indicate that CCR5 oligomers are involved in an internalization process that is distinct from that induced by the receptor's agonists.
Blanpain
Cédric
C
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, B-1070 Brussels, Belgium.
Vanderwinden
Jean-Marie
JM
Cihak
Josef
J
Wittamer
Valérie
V
Le Poul
Emmanuel
E
Issafras
Hassan
H
Stangassinger
Manfred
M
Vassart
Gilbert
G
Marullo
Stefano
S
Schlndorff
Detlef
D
Parmentier
Marc
M
Mack
Matthias
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D065100:CCR5 Receptor Antagonists; D006224:Cricetinae; D004705:Endocytosis; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary / Q000502:physiology; D019713:Receptors, CCR5 / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction; D014779:Virus Replication
D000911:Antibodies, Monoclonal; D065100:CCR5 Receptor Antagonists; D019713:Receptors, CCR5