Mutations of serine 236-237 and tyrosine 302 residues in the human lipoxin A4 receptor intracellular domains result in sustained signaling
Mutations of serine 236-237 and tyrosine 302 residues in the human lipoxin A4 receptor intracellular domains result in sustained signaling
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DOI：
10.1021/bi001196i
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发表时间：
2000-11-07
期刊：
影响因子：
2.9
通讯作者：
Fiore, S
中科院分区：
文献类型：
--
作者：
Kang, Y;Taddeo, B;Fiore, S
脂素A(4) （LXA(4)）是一种有效的炎症反应负调节因子。LXA(4)的抗炎活性，如抑制激动剂诱导的多形核细胞（PMN）趋化和上调β -2整合素，需要g蛋白偶联的高亲和力LXA(4)受体（LXA(4)R）的表达。我们现在报道了促炎激动剂n -甲酰基肽（FMLP）和钙离子载体A对PMN的刺激（23187）。随后LXA(4)结合明显下调（B-max降低45%），磷脂酶A(2) （PLA(2)）和D （PLD）的激活降低。通过对LXA(4)R细胞内结构域的结构功能分析，阐明了这些作用的机制。突变分子S236/5237 -> A/G (LXA（4)R(pk)）和Y302 -> F (LXA（4)R(tk)）通过定点诱变获得，产生缺乏丝氨酸/苏氨酸激酶或酪氨酸激酶依赖性磷酸化的假定靶点的受体。利用野生型和突变的LXA(4)R序列在CHO和HL-60细胞中的表达来检测LXA(4)配体-受体相互作用和信号转导事件。结果表明，表达LXA(4)R（pk）或LXA(4)R（tk）的细胞表现出PLA(2)和PLD的持续激活，而表达LXA(4)R（wt）的细胞则表现出短暂的PLA（2 -3 min的峰值激活）。此外，在表达LXA(4)R（pk）的细胞中，未观察到PMA对LXA(4)R（wt）转染的CHO细胞中LXA(4)R依赖性PLA2活性的抑制作用。磷酸化免疫印迹显示野生型和突变型LXA(4)受体之间的功能差异伴随着受体蛋白磷酸化模式的明显变化。进一步表征这些和相关的LXA(4)R细胞内结构域将有助于更好地理解调节LXA抗炎活性的特定事件(4)。
Lipoxin A(4) (LXA(4)) is a potent negative modulator of the inflammatory response. The antiinflammatory activities of LXA(4), such as inhibition of agonist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of beta -2 integrins, require the expression of a G-protein-coupled, high-affinity LXA(4) receptor (LXA(4)R). We now report that stimulation of PMN with proinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A(23187). or phorbol mirystate acetate (PMA) is followed by marked downregulation of LXA(4) binding (B-max decrease of similar to 45%) and decreased activation of phospholipases A(2) (PLA(2)) and D (PLD). Elucidation of the mechanisms underlying these effects was addressed by structure-function analyses of the intracellular domains of LXA(4)R. Mutant molecule, S236/5237 -> A/G (LXA(4)R(pk)) and Y302 -> F (LXA(4)R(tk)) were obtained by site-directed mutagenesis to yield receptors lacking the putative targets fur serine/threonine kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-type and mutated LXA(4)R sequences in CHO and HL-60 cells was used to examine LXA(4) ligand-receptor interactions and signal transduction events. Results indicated that cells expressing LXA(4)R(pk) or LXA(4)R(tk) displayed sustained activation of PLA(2) and PLD in contrast to the transient ones obtained with LXA(4)R(wt) (peak activation at 2-3 min). Moreover, inhibition of LXA(4)-dependent PLA2 activity by PMA in LXA(4)R(wt) transfected CHO cells was not observed in cells expressing LXA(4)R(pk). Phosphopeptide immunoblotting revealed that the functional differences between wild-type and mutant LXA(4) receptors are accompanied by distinct changes in the receptor protein phosphorylation pattern. Further characterization of these and related LXA(4)R intracellular domains will help to better understand specific events that regulate the antiinflammatory activities of LXA(4).