Lymphotoxin β receptor (LTβR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTβR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTβR internalization to its signaling potential. Intracellular localization of LTβR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTβR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTβR stimulation was assessed by qRT-PCR analysis. We demonstrated that LTβR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTβR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTβR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTβR-triggered activation of the non-canonical branch of the NF-κB pathway. Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTβR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli.

中文翻译：

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网格蛋白和动力蛋白依赖性内吞作用限制了淋巴毒素 β 受体触发的经典 NF-κB 信号传导

淋巴毒素 β 受体 (LTβR) 是肿瘤坏死因子受体 (TNFR) 超家族的成员，可调节免疫反应。在细胞水平上，在配体结合后，受体激活促炎性 NF-κB 和 AP-1 通路。然而，LTβR 的细胞内分布、其内吞作用的途径及其与信号激活的联系尚未得到表征。在这里，我们研究了 LTβR 内化对其信号传导潜力的贡献。通过共聚焦显微镜分析未受刺激和受刺激细胞中 LTβR 的细胞内定位。内吞损伤是通过 siRNA 或 CRISPR/Cas9 介导的消耗或化学抑制调节内吞途径的蛋白质来实现的。检查了 LTβR 诱导的信号传导的激活。通过蛋白质印迹法测量了 NF-κB 通路经典和非经典分支的效应蛋白水平，以及参与不同信号级联反应的 JNK、Akt、ERK1/2、STAT1 和 STAT3 的磷酸化水平。通过 qRT-PCR 分析评估对 LTβR 刺激的转录反应。我们证明 LTβR 主要存在于内吞囊泡和高尔基体上。受体的配体结合池定位于内体并被运送到溶酶体进行降解。不同内吞途径（网格蛋白介导的、动力蛋白依赖性或网格蛋白非依赖性的）调节因子的消耗导致 LTβR 内化受损，表明该受体使用多种进入途径。缺乏网格蛋白和动力蛋白的细胞表现出增强的经典 NF-κB 信号激活，表现为 IκBα 抑制剂降解增加和 LTβR 靶基因表达升高。我们还证明网格蛋白和动力蛋白缺乏在一定程度上减少了 LTβR 触发的 NF-κB 途径非规范分支的激活。我们的工作表明网格蛋白和动力蛋白依赖性内化的损害放大了细胞对 LTβR 刺激的反应。我们假设受体内化限制了细胞对阈下刺激的反应。我们的工作表明网格蛋白和动力蛋白依赖性内化的损害放大了细胞对 LTβR 刺激的反应。我们假设受体内化限制了细胞对阈下刺激的反应。我们的工作表明网格蛋白和动力蛋白依赖性内化的损害放大了细胞对 LTβR 刺激的反应。我们假设受体内化限制了细胞对阈下刺激的反应。