ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

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 Ca sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca remains unclear. Here, we find that both intracellular and extracellular Ca 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 Ca pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca, 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 Ca oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca dynamics.