Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.
Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.
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DOI：
10.4049/jimmunol.151.11.6418
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发表时间：
1993-12
影响因子：
4.4
通讯作者：
I. Schraufstatter;D. Barritt;M. Ma;Z. Oades;C. Cochrane
中科院分区：
文献类型：
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作者：
I. Schraufstatter;D. Barritt;M. Ma;Z. Oades;C. Cochrane
为了确定IL-8与其两种已知受体结合的重要结构特征，从pGEX 2T大肠杆菌表达载体中构建并纯化了IL-8和黑素瘤生长刺激活性（MGSA）的突变体和由这两种趋化因子的片段组成的嵌合体。IL-8 α和β受体在293肾上皮细胞和HL 60人白血病细胞中稳定且单独地表达。IL-8本身的Kd和转染细胞中两种受体的拷贝数相当。然而，与125I标记的IL-8的竞争结合显示出α和β受体之间的几种IL-8突变体的巨大差异。氨基末端ELR序列对于IL-8与α受体的结合是重要的，但对于高亲和力结合是不够的。兔IL-8和MGSA都与人IL-8共享ELR序列，但与之竞争性差。兔IL-8/人IL-8嵌合体与人IL-8序列仅在8个氨基酸上不同，其对α受体的亲和力比人IL-8低150倍。相比之下，氨基和羧基末端似乎对于与β受体结合是重要的。如果IL-8的ELR序列被丙氨酸取代，或者如果C50远端的羧基末端被MGSA序列取代，则结合竞争减少。如果这两项修改同时进行，则约束力被废除。通过用丙氨酸替换ELR序列完全阻止MGSA的结合。用α或β受体转染的HL 60细胞中的Ca2+动员用于评估细胞刺激。IL-8的各种突变形式诱导的受体活性与竞争结合亲和力平行的敏感性模式，表明两种受体都是活性的。
To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.