Pleckstrin homology domain-containing family G member 4B
cell-cell junction
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 Ca<sup>2+</sup> 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, Ca<sup>2+</sup> influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca<sup>2+</sup> or BAPTA-AM (an intracellular Ca<sup>2+</sup> 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)P<sub>2</sub>] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca<sup>2+</sup> levels and PI(4,5)P<sub>2</sub> 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 Ca<sup>2+</sup> influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.