Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4
Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4
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DOI：
10.1111/j.1349-7006.2007.00388.x
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发表时间：
2007-03-01
期刊：
影响因子：
5.7
通讯作者：
Yamamoto, Naoki
中科院分区：
文献类型：
--
作者：
Futahashi, Yuko;Komano, Jun;Yamamoto, Naoki
转移增强剂 CXCR4 的 C 端胞质结构域调节其功能和时空表达。然而，与 CXCR4 的配体基质细胞衍生因子 1 α (SDF-1 α)/CXCL12 介导的内化相比，我们对 CXCR4 组成性内化的机制知之甚少。我们建立了一个系统，使用缺乏内源 CXCR4 和 SDF-1 α 的 NP2 细胞系来分析 CXCR4 细胞质尾在稳态内化中的作用。从 C 末端删除超过 6 个氨基酸会显着减少 CXCR4 的组成型内化。丙氨酸取代突变揭示其中三个氨基酸 Ser(344) Glu(345) Ser(346) 对于 CXCR4 的有效稳态内化至关重要。将 Glu(345) 突变为 Asp 不会破坏内化，表明稳态内化基序是 S(E/D)S。当测试对SDF-1α的反应时，表达缺乏C端10、14、22、31或44个氨基酸的CXCR4突变体的细胞没有表现出细胞表面CXCR4的下调或SDF-1α诱导的细胞迁移。然而有趣的是，我们鉴定了两种突变体，一种具有 E344A 突变，另一种缺乏 C 端 17 个氨基酸，它们在组成性内化方面存在缺陷，但在配体促进的内化和细胞迁移方面具有能力。这些数据表明，配体依赖性和非依赖性内化在遗传上是可分离的，并且在氨基酸336和342之间，存在配体促进内化的负调节元件。讨论了这个新基序在癌症转移和其他 CXCR4 相关疾病（如疣、低丙种球蛋白血症、感染和骨髓缺乏症 (WHIM) 综合征）中的潜在参与。
The C-terminal cytoplasmic domain of the metastatic potentiator CXCR4 regulates its function and spatiotemporal expression. However, little is known about the mechanism underlying constitutive internalization of CXCR4 compared to internalization mediated by its ligand, stromal cell-derived factor-1 alpha (SDF-1 alpha)/CXCL12. We established a system to analyze the role of the CXCR4 cytoplasmic tail in steady-state internalization using the NP2 cell line, which lacks endogenous CXCR4 and SDF-1 alpha. Deleting more than six amino acids from the C-terminus dramatically reduced constitutive internalization of CXCR4. Alanine substitution mutations revealed that three of those amino acids Ser(344) Glu(345) Ser(346) are essential for efficient steady-state internalization of CXCR4. Mutating Glu(345) to Asp did not disrupt internalization, suggesting that the steady-state internalization motif is S(E/D)S. When responses to SDF-1 alpha were tested, cells expressing CXCR4 mutants lacking the C-terminal 10, 14, 22, 31 or 44 amino acids did not show downregulation of cell surface CXCR4 or the cell migration induced by SDF-1 alpha. Interestingly, however, we identified two mutants, one with E344A mutation and the other lacking the C-terminal 17 amino acids, that were defective in constitutive internalization but competent in ligand-promoted internalization and cell migration. These data demonstrate that ligand-dependent and -independent internalization is genetically separable and that, between amino acids 336 and 342, there is a negative regulatory element for ligand-promoted internalization. Potential involvement of this novel motif in cancer metastasis and other CXCR4-associated disorders such as warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome is discussed.