Pediatric Hematology-Oncology, University of California, San Diego, La Jolla, Calif. 92017, USA.
Exp Hematol. 2010 May;38(5):384-91. doi: 10.1016/j.exphem.2010.02.007. Epub 2010 Feb 24.
To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists.
We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species.
Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin.
These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.
确定特定的 c-Mpl 突变是否可能对血小板生成素受体激动剂产生反应。
我们创建了在先天性巨核细胞血小板减少症中发现的 II 型 c-Mpl 突变的细胞系模型。我们选择 F104S c-Mpl 进行进一步研究，因为它表现出受体的表面表达。我们通过 1-甲基四唑-5-硫醇测定法测量表达野生型或 F104S c-Mpl 的细胞系对靶向受体细胞外（m-AMP4）或跨膜（LGD-4665）结构域的血小板生成素受体激动剂的增殖反应。我们使用体外血小板生成素摄取测定法测量突变受体与血小板生成素的结合，并通过对齐来自多种物种的血小板生成素和促红细胞生成素受体，确定 F104 是受体与其配体相互作用的潜在关键残基。
表达 F104S c-Mpl 的细胞对 LGD-4665 增殖反应，但对血小板生成素或 m-AMP4 无反应。与血小板生成素相比，LGD-4665 在表达野生型和 F104S c-Mpl 的细胞中刺激信号传递具有延迟的动力学。尽管 F104S c-Mpl 在我们的 BaF3 细胞系模型中表达在细胞表面上，但突变受体不与血小板生成素结合。与促红细胞生成素受体相比，这表明 F104 参与氢键相互作用，这对于与血小板生成素结合至关重要。
这些发现表明，一小部分先天性巨核细胞血小板减少症患者可能对血小板生成素受体激动剂的治疗有反应，但反应性将取决于突变类型和激动剂的使用。我们推测 F104 对于血小板生成素的结合至关重要。与血小板生成素相比，对跨膜结构域结合激动剂的信号传递动力学延迟。
