Fiore S, Serhan C N
Department of Anesthesia, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Biochemistry. 1995 Dec 26;34(51):16678-86. doi: 10.1021/bi00051a016.
Lipoxin A4 (LXA4) interacts with high-affinity receptors in human neutrophils and differentiated HL-60 cells. Recently, we characterized a myeloid-derived cDNA that encodes a LXA4 high-affinity receptor (LXA4R) [Fiore, S., Maddox, J. F., Perez, H. D., and Serhan, C. N. (1994) J. Exp. Med. 180, 253-260] denoted earlier as a related N-formyl peptide receptor (RFP). To examine the selectivity of this receptor we tested its preference for specific binding of 3H-LXA4 versus 3H-N-formylmethionyl-leucyl-phenylalanine (3H-FMLP). When receptor-transfected Chinese hamster ovary cells were exposed to either 3H-LXA4 or 3H-FMLP, the receptor affinity for LXA4 exceeded by 1000-fold that of FMLP (6.1 nM vs 5 microM). Upon differentiation, HL-60 cells acquire high-affinity binding sites and respond to both LXA4 and FMLP. Northern blot analysis of differentiated HL-60 cells using an RFP probe showed a characteristic band at 2.1 kb. Differentiated HL-60 cells exposed to an RFP antisense oligonucleotide selectively lost 3H-LXA4 binding as well as LXA4-stimulated lipid remodeling that paralleled the loss of mRNA for LXA4R. In contrast, the specific mRNA for the FMLP receptor, 3H-FMLP specific binding, and FMLP-induced phospholipase D activity were still observed. Treatment of human neutrophils with antisera raised against a peptide in the LXA4R third extracellular domain also resulted in selective abrogation of 3H-LXA4 specific binding with polymorphonuclear leukocytes (PMN) without blocking 3H-FMLP binding. FMLP-stimulated CD11b upregulation as well as homotypic aggregation of PMN was inhibited by LXA4 (which at 10(-9) M gave approximately 1 log unit shift to the right in the FMLP dose-response curve). The addition of LXA4R antisera did not alter FMLP-induced responses in PMN but completely blocked LXA4 actions. These results indicate that altering the expression of the LXA4R protein by blockage of transcriptional mechanisms or hindrance of the LXA4R extracellular domains leads to loss of LXA4 specific binding and blockage of LXA4 signaling. Moreover, they indicate that in myeloid cells LXA4-LXA4R interactions are dissociable from those of FMLP and that LXA4 regulates CD11/18 on the PMN surface.
脂氧素A4（LXA4）与人中性粒细胞及分化的HL-60细胞中的高亲和力受体相互作用。最近，我们鉴定了一种髓系来源的cDNA，其编码一种LXA4高亲和力受体（LXA4R）[菲奥雷，S.，马多克斯，J. F.，佩雷斯，H. D.，和塞尔汉，C. N.（1994年）《实验医学杂志》180，253 - 260]，该受体先前被称为相关N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸受体（RFP）。为了检测该受体的选择性，我们测试了其对3H-LXA4与3H-N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（3H-FMLP）特异性结合的偏好性。当将受体转染的中国仓鼠卵巢细胞暴露于3H-LXA4或3H-FMLP时，该受体对LXA4的亲和力比对FMLP的亲和力高1000倍（6.1 nM对5 μM）。分化后，HL-60细胞获得高亲和力结合位点，并对LXA4和FMLP均有反应。使用RFP探针对分化的HL-60细胞进行Northern印迹分析，显示在2.1 kb处有一条特征带。暴露于RFP反义寡核苷酸的分化HL-60细胞选择性地丧失了3H-LXA4结合以及LXA4刺激的脂质重塑，这与LXA4R mRNA的丧失平行。相反，仍可观察到FMLP受体的特异性mRNA、3H-FMLP特异性结合以及FMLP诱导的磷脂酶D活性。用针对LXA4R第三细胞外结构域中一种肽产生的抗血清处理人中性粒细胞，也导致多形核白细胞（PMN）对3H-LXA4特异性结合的选择性消除，而不阻断3H-FMLP结合。LXA4抑制FMLP刺激的CD11b上调以及PMN的同型聚集（10^(-9) M的LXA4使FMLP剂量反应曲线向右移动约1个对数单位）。添加LXA4R抗血清不会改变FMLP诱导的PMN反应，但完全阻断LXA4的作用。这些结果表明，通过阻断转录机制或阻碍LXA4R细胞外结构域来改变LXA4R蛋白的表达，会导致LXA4特异性结合的丧失以及LXA4信号传导的阻断。此外，它们表明在髓系细胞中，LXA4与LXA4R的相互作用与FMLP的相互作用是可分离的，并且LXA4调节PMN表面的CD11/18。
