Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies
Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies
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DOI：
10.1091/mbc.01-03-0129
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发表时间：
2002-02-01
影响因子：
3.3
通讯作者：
Mack, M
中科院分区：
文献类型：
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作者：
Blanpain, C;Vanderwinden, JM;Mack, M
CC 趋化因子受体 5 (CCR5) 是人类免疫缺陷病毒 1 型 (HIV-1) 嗜巨噬细胞株的主要辅助受体。我们生成了一组抗 CCR5 单克隆抗体，并在表位识别、与趋化因子结合的竞争、受体激活和运输以及辅助受体活性方面对它们进行了表征。 MC-4、MC-5 和 MC-7 映射到氨基末端结构域，MC-1 映射到第二个细胞外环，MC-6 映射到覆盖多个细胞外结构域的构象表位。 MC-1和MC-6抑制正常T细胞表达和分泌的活化调节(RANTES)、巨噬细胞炎症多肽-1β和Env结合，而MC-5抑制巨噬细胞炎症多肽-1β和Env但不抑制RANTES结合。 MC-6 在不同的功能测定中诱导信号传导，表明这种单克隆抗体稳定了 CCR5 的活性构象。流式细胞术和实时共聚焦显微镜显示MC-1促进了强烈的CCR5内吞作用。 MC-I而非其单价异构体诱导CCR5分子之间的能量转移增加。此外，其单价异构体可以有效结合，但不会内化受体。相反，MC-4 不会阻止 RANTES 结合或随后的信号传导，但抑制其促进 CCR5 内化的能力。这些结果表明CCR5存在多种活性构象，并表明CCR5寡聚物参与了与受体激动剂诱导的内化过程不同的内化过程。
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-I and MC-6 inhibited reculated 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-I 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.