Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Dev Comp Immunol. 2012 Jan;36(1):62-73. doi: 10.1016/j.dci.2011.06.004. Epub 2011 Jun 15.
Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.
由各种髓样和淋巴样免疫细胞亚群表达的鲫鱼（Ictalurus punctatus）白细胞免疫型受体（IpLITR）被预测在启动和终止硬骨鱼细胞效应反应中发挥关键作用。这些 I 型跨膜蛋白属于免疫球蛋白超家族，具有免疫调节受体的特征，具有抑制和/或刺激信号潜能。在我们之前的工作基础上，该工作表明与鱼的衔接蛋白 IpFcRγ 和 FcRγ-L 相关的假定刺激型 IpLITR 型，本研究集中于该免疫受体衔接子信号复合物的功能意义。具体而言，我们通过将 IpLITR 2.6b 的细胞外结构域与 IpFcRγ-L 的跨膜区和胞质尾融合，生成了一个表位标记的嵌合受体构建体。该嵌合体在大鼠嗜碱性白血病（RBL）细胞系 RBL-2H3 中稳定表达，并且在表面受体与抗血凝素单克隆抗体或调理微球交联后，嵌合硬骨鱼受体分别诱导细胞脱颗粒和吞噬反应。在嵌合体胞质尾中编码的免疫受体酪氨酸基激活基序的定点突变证实，这些功能反应依赖于该基序中磷酸化的酪氨酸。使用磷酸特异性抗体和药理学抑制剂的组合，我们还证明 IpLITR/IpFcRγ-L 诱导的脱颗粒反应需要 SH2 结构域含有蛋白酪氨酸磷酸酶、磷脂酰肌醇 3-激酶、蛋白激酶 C 和丝裂原活化蛋白激酶的活性，但似乎独立于 c-Jun N-末端激酶和 p38 MAP 激酶途径。除了首次观察到刺激型 IpLITR 介导的信号及其对细胞效应反应的影响外，还将讨论生成稳定表达功能性 IpLITR-衔接子嵌合体的 RBL-2H3 细胞的优势。
