The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by siglecs-7 and-9, CD33-related Siglecs expressed on human monocytes and NK cells
The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by siglecs-7 and-9, CD33-related Siglecs expressed on human monocytes and NK cells
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DOI：
10.4049/jimmunol.173.11.6841
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发表时间：
2004-12-01
影响因子：
4.4
通讯作者：
Crocker, PR
中科院分区：
文献类型：
--
作者：
Avril, T;Floyd, H;Crocker, PR
Siglec-7和Siglec-9是最近表征的唾液酸结合蛋白的CD 33相关Siglec家族的两个成员，并且都在人单核细胞和NK细胞上表达。除了它们识别唾液酸残基的能力之外，这些Siglecs在它们的细胞质区域中显示两个保守的基于酪氨酸的基序，其类似于在免疫系统的抑制性受体中发现的那些基序。在本研究中，我们使用大鼠嗜碱性白血病（RBL）模型来检查Siglecs-7和-9作为抑制性受体发挥作用的潜力，并研究其分子基础。我们首先证明了Siglecs-7和Siglecs-9能够在共交联后抑制Fc ε RI介导的5-羟色胺从RBL细胞释放。此外，我们表明，在这些条件下或过钒酸盐处理后，Siglecs-7和-9与Src同源区2域含有磷酸酶（SHP），SHP-1和SHP-2，无论是在免疫沉淀和荧光显微镜实验中使用GFP融合蛋白。然后，我们通过定点诱变表明，近膜酪氨酸基序对于Siglec-7和Siglec-9的抑制功能是必不可少的，并且也是酪氨酸磷酸化和SHP-1和SHP-2磷酸酶募集所必需的。最后，近膜基序的突变增加了Siglecs-7和Siglecs-9的唾液酸结合活性，提高了可能发生“由内而外”信号传导以调节配体结合的可能性。
Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.