Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits
Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits
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DOI：
10.1021/bi061108f
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发表时间：
2006-12-26
期刊：
影响因子：
2.9
通讯作者：
Stern, Lawrence J.
中科院分区：
文献类型：
--
作者：
Sigalov, Alexander B.;Aivazian, Dikran A.;Stern, Lawrence J.
T细胞、B细胞、肥大细胞、自然杀伤细胞、嗜碱性粒细胞和其他免疫细胞表面的多链免疫识别受体(MIRR)是由几种单一的跨膜蛋白结合而成的，免疫球蛋白样配体识别结构域和信号转导结构域存在于不同的亚基上。MIRR信号转导亚单位都含有一个或多个基于免疫受体酪氨酸激活基序(ITAM)的拷贝的细胞质结构域，当受体参与信号级联的早期和必需事件时，ITAM的酪氨酸残基被磷酸化。尽管ITAM靠近细胞膜，在跨膜信号转导中起着重要作用，但人们对含ITAM的细胞质结构域的结构和脂质结合活性知之甚少。在这里，我们用纯化的重组蛋白研究了几个MirR胞质结构域，即T细胞受体Zeta(Cyt)、CD3 epsilon(Cyt)、CD3 Delta(Cyt)和CD3 Gamma(Cyt)、B细胞受体Igα(Cyt)和Igβ(Cyt)以及Fc受体Fc epsilon RI Gamma(Cyt)的构象和脂质结合活性。二级结构预测分析和实验圆二色谱确定这些细胞质结构域中的每一个都是天然未折叠的蛋白质。我们还报道了Zeta(Cyt)、CD3 epsilon(Cyt)和Fc epsilon RI Gamma(Cyt)与酸性和混合磷脂小泡结合，结合强度与蛋白质净电荷和聚集的碱性氨基酸残基的存在有关。圆二色谱分析表明，这些结构域缺乏脂质结合形式的二级结构。Zeta(Cyt)和Fc epsilon RI Gamma(Cyt)的磷酸化不会改变它们的无规卷曲构象，但会削弱与膜的结合。讨论了这些结果对免疫受体跨膜信号转导的意义。
Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely, T cell receptor zeta(cyt), CD3 epsilon(cyt), CD3 delta(cyt), and CD3 gamma(cyt), B cell receptor Ig alpha(cyt) and Ig beta(cyt), and Fc receptor Fc epsilon RI gamma(cyt), using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that zeta(cyt), CD3 epsilon(cyt), and Fc epsilon RI gamma(cyt) bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of zeta(cyt) and Fc epsilon RI gamma(cyt) does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.