The microtubule-binding protein hook3 interacts with a cytoplasmic domain of scavenger receptor A
The microtubule-binding protein hook3 interacts with a cytoplasmic domain of scavenger receptor A
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DOI：
10.1074/jbc.m611537200
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发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Kuroki, Yoshio
中科院分区：
文献类型：
--
作者：
Sano, Hitomi;Ishino, Masaho;Kuroki, Yoshio
A类清道夫受体（SR-A）是一种多功能跨膜糖蛋白，参与动脉粥样硬化形成、先天免疫和细胞粘附。尽管对受体的结构-功能进行了广泛的研究，但直接与SR-A相互作用并调节受体运输的细胞内分子尚未确定。在目前的研究中，我们已经确定了一个微管结合蛋白，钩3，作为一个新的相互作用的合作伙伴SR-A。通过酵母双杂交筛选和大鼠肺泡巨噬细胞中SR-A-胞质结构域结合蛋白的质谱分析，表明大鼠Hook3亚型与SR-A之间存在关联。通过下拉测定和免疫共沉淀证明过表达和内源性人Hook3与SR-A的结合。此外，内源性鼠SR-A和HK3从分离自Raw 264.7鼠巨噬细胞的细胞裂解物中共沉淀。SR-A识别细胞外配体后，Hook3与SR-A的相互作用被显著激活。使用截短的研究表明，人Hook 3的带正电荷的C-末端瓦尔（614）-Ala（717）区域是与人SR-A胞质结构域中带负电荷的残基Glu（12）、Asp（13）和Asp（15）相互作用所必需的。通过干扰Hook3的小干扰RNA，SR-A的总表达和表面表达、受体介导的配体摄取和蛋白稳定性显著提高，而蛋白合成和成熟没有改变。我们首次提出Hook3可能参与内吞清道夫受体的周转。
The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellar molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val(614)-Ala(717) region of human Hook3 was required for the interaction with the negatively charged residues, Glu(12), Asp(13), and Asp(15) in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.