Abstract：
Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.
摘要：
巨细胞胞吞作用（MPC）是一种大规模的内吞作用途径，涉及肌动蛋白依赖性膜皱褶的形成和随后的皱褶闭合，以产生用于摄取液相货物的巨细胞胞体。MPC分为两种类型：组成型和刺激诱导型。巨噬细胞中的组成型MPC依赖于钙感应受体的细胞外Ca2感应。然而,刺激诱导的MPC与Ca2+之间的联系尚不清楚。这里,我们发现A431人表皮样癌细胞中表皮生长因子（EGF）诱导的MPC需要细胞内和细胞外Ca2。通过研究腔体细胞摄取缺陷（CUP）基因的哺乳动物同源物，我们鉴定了ATP2B4，编码称为质膜钙ATP酶4（PMCA4）的Ca2+泵，作为EGF诱导的MPC的Ca2相关调节因子。ATP2B4的敲除（KO），以及细胞外/细胞内Ca2+的消耗，抑制荷叶边闭合和macropinosome形成，不影响荷叶边的形成。我们证明了PMCA4活性本身的重要性，独立于通过其C末端与其他蛋白质的相互作用，称为PDZ结构域结合基序。此外,我们表明ATP2B4-KO降低了MPC过程中EGF刺激的Ca2振荡。我们的发现表明EGF诱导的MPC需要ATP2B4依赖性Ca2动力学。
