CXCR1 and CXCR2 activation and regulation - Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization
CXCR1 and CXCR2 activation and regulation - Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization
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DOI：
10.1074/jbc.m610289200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Richardson, Ricardo M.
中科院分区：
文献类型：
--
作者：
Nasser, Mohd W.;Raghuwanshi, Sandeep K.;Richardson, Ricardo M.
CXCL 8（白细胞介素-8）与两种受体CXCR 1和CXCR 2相互作用，以激活白细胞。在活化后，CXCR 2相对于CXCR 1非常迅速地内化（5分钟后类似于90%对比类似于10%）。受体的C末端已被证明是必需的内化，但不足以解释不同的动力学下调。为了确定调节受体内化的结构决定簇，通过在CXCR 1和CXCR 2之间逐渐交换特定结构域或氨基酸来产生各种嵌合和点突变受体。该受体在大鼠嗜碱性白血病2 H3细胞中稳定表达，并表征为受体结合、细胞内Ca 2+动员、磷酸肌醇水解、磷酸化、内化和MAPK活化。本文的数据表明，受体的第二胞外环（2 ECL）对于不同的内化速率至关重要。用CXCR 1的2 ECL（B（2 ECL）A）或用CXCR 1的缬氨酸对应物（B（D199 V）A）替换CXCR 2的2 ECL，与CXCR 1类似，延迟了CXCR 2的内化。用Asn（B-D199 N）替换Asp（199）恢复了CXCR 2的快速内化。受体的2 ECL的结构建模也表明，Asp（199）在稳定和调节CXCR 2相对于CXCR 1的快速内化中起关键作用。BD 199 N迅速内化，但作为单一磷酸化形式迁移，如CXCR 1（类似于75 kDa），而B（2 ECL）A和B（D199 V）A显示缓慢和快速迁移形式，如CXCR 2（分别类似于45和65 kDa），但内化，如CXCR 1。这些数据进一步削弱了受体寡聚化在CXCL 8受体内化中的作用。与CXCR 1类似，相对于BD 199 N和CXCR 2，B（D199 V）A也诱导持续的ERK激活和交叉脱敏的Ca 2+向CCR 5的动员。总之，这些数据表明，CXCL 8受体的2 ECL在调节其不同的下调速率，从而调节信号长度和内化后活性方面是重要的。
CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (similar to 90% versus similar to 10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca2+ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B(2ECL)A) or Asp(199) with its CXCR1 valine counterpart (B(D199V)A) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp(199) with Asn (B-D199N) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp(199) plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. BD199N internalized rapidly but migrated as a single phosphorylated form like CXCR1 (similar to 75 kDa), whereas B(2ECL)A and B(D199V)A showed slow and fast migrating forms like CXCR2 (similar to 45 and similar to 65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B(D199V)A also induced sustained ERK activation and cross-desensitized Ca2+ mobilization to CCR5 relative to BD199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.