Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2
Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2
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DOI：
10.1074/jbc.m303739200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Peiper, SC
中科院分区：
文献类型：
--
作者：
Arias, DA;Navenot, JM;Peiper, SC
CCR 5是RANTES、MIP-1 α、MIP-1 β和MCP-2的G蛋白偶联受体，作为人类免疫缺陷病毒1型感染的前线辅助受体发挥作用。为了阐明CCR 5激活的机制，这种辅助受体在酵母中表达耦合到信息素反应途径和组成型活性突变体（CAM）的随机诱变。跨膜螺旋2中高度保守的TXP基序中的Thr-82转化为Pro、His、Tyr、Arg或Lys在酵母和哺乳动物细胞中赋予自主信号传导活性。这种取代也赋予了酵母和哺乳动物细胞中的CCR 2组成型信号传导，而不是CCR 1、CCR 3、CCR 4、CXCR 2或CXCR 4。CCR 5-CAM而非CCR 2-CAM具有配体结合亲和力的降低。而RANTES刺激诱导的钙动员幅度在CCR 5-CAM中低于野生型（WT）受体，MCP-1在CCR 2-CAM中诱导的信号高于CCR 2-WT。CCR 5-CAM（T82 P）对RANTES的趋化反应与CCR 5-WT相似，但CCR 5-CAM（T82 K）对RANTES的趋化反应显著降低。CCR 2-WT和CCR 2-CAM（T94 K）的趋化反应相似。这些发现扩展了对TXP基序在CCR 5信号传导机制中的作用的了解。CCR 2是与CCR 5遗传关系最密切的受体，具有相似的信号传导机制，但其他含有TXP基序的受体则没有。CCR 5和CCR 2在酵母中的表达以及具有自主信号传导的变体的可用性代表了表征受体拮抗剂和开发阻断其在人类疾病中的作用的方法的关键工具。
CCR5 is a G protein-coupled receptor for RANTES, MIP-1alpha, MIP-1beta, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.