Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor α2 chain
Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor α2 chain
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DOI：
10.1074/jbc.m100936200
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发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Puri, RK
中科院分区：
文献类型：
--
作者：
Kawakami, K;Takeshita, F;Puri, RK
白细胞介素（IL）-13受体α 2（IL-13 R α 2）链是IL-13介导的配体结合的必需结合组分。最近，我们已经证明，这种受体链也发挥了重要作用，在IL-13的内化。为了研究IL-13内化的机制，我们产生了突变的IL-13 R α 2链，其靶向跨膜结构域中的三亮氨酸残基（Leu（335）、Leu（336）和Leu（337））和胞内结构域中的酪氨酸基序（Tyr（343）），并将这些cDNA转染COS-7细胞。表达C末端截短的IL-13 R α 2链（Delta 335）的细胞不结合IL-13，表明三亮氨酸区域调节IL-13结合。与野生型IL-13 R α 2链转染的细胞相比，在三亮氨酸区具有突变的IL-13 R α 2链的截短导致内化显著降低。与野生型IL-13 R α 2链转染的细胞相比，用酪氨酸基序突变体转染的COS-7细胞表现出相似的内化水平;然而，与野生型IL-13 R α 2转染子相比，细胞表面IL-13的解离更快。通过测定由IL-13和突变形式的假单胞菌外毒素（IL-13-PE 38 QQR）组成的嵌合蛋白对表达IL-13 R α 2链突变体的细胞的细胞毒性，进一步证实了这些结果。我们进一步证明IL-13 R α 2链不是泛素化的，IL-13 R α 2的内化不依赖于泛素化。总之，我们的研究结果表明，三亮氨酸区的双亮氨酸基序和酪氨酸基序以不同的方式参与IL-13 R α 2的内化。
Interleukin (IL)-13 receptor alpha2 (IL-13R alpha2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13R alpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13R alpha2 chain (Delta 335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13R alpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13R alpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13R alpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13R alpha2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13R alpha2 chain mutants. We further demonstrate that the IL-13R alpha2 chain is not ubiquitinated and that internalization of IL-13R alpha2 did not depend on ubiquitination, Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13R alpha2 internalization in distinct manners.