Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal CA²⁺ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (AnxA2)-binding ability. Thus, CA²⁺ influx and AnxA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low CA²⁺ or BAPTA-AM (an intracellular CA²⁺ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P₂] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular CA²⁺ levels and PI(4,5)P₂ binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that CA²⁺ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process. [Media: see text] [Media: see text].