Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region
Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region
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DOI：
10.4049/jimmunol.165.9.5295
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发表时间：
2000-11-01
影响因子：
4.4
通讯作者：
Kolattukudy, PE
中科院分区：
文献类型：
--
作者：
Preobrazhensky, AA;Dragan, S;Kolattukudy, PE
单核细胞趋化蛋白-1(MCP-1)与其受体CCR2B结合，在感染、炎症和/或损伤等多种疾病中发挥重要作用。为了了解这种相互作用的分子基础及其生物学后果，我们在包括CCR2B在内的几个趋化因子受体的N端胞外区域发现了一个保守的十六位氨基酸。表达FLAG标记的CCR2B的人胚胎肾脏293细胞包含该区域21-26的定点突变，包括一个共同的酪氨酸硫化位点，用于确定MCP-1的结合及其生物学意义。结果表明，这些氨基酸中的几个对MCP-1结合和随后的片层脂层的形成、趋化和涉及腺苷环化酶抑制和钙内流的信号转导是重要的，阻止腺苷环化酶抑制和钙内流的突变并不显著地抑制片层脂层的形成和趋化，这表明这些信号事件不参与趋化作用，CCR2B被发现在Tyr(26)处被硫酸盐化；这种硫化被Ala取代Tyr而被Ala取代，并被普遍认为的硫酸盐化位点之一Asp(25)取代而严重减少。表达的CCR2B被发现是N-糖基化的，因为受体的N-糖苷酶F处理或细胞在衣霉素中的生长将受体的大小减少到相同的水平，从50 kDa到45 kDa。因此，CCR2B是CC趋化因子受体家族的第一个成员，被证明是一种糖蛋白，在N端的Tyr处被硫酸盐化。这些修饰可能具有重要的生物学功能。
Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21-26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm, Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis, CCR2B was found to be sulfated at Tyr(26); this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp(25), a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These modifications probably have significant biological functions.