End binding protein 3 promotes IP3‐gated calcium release and permeability of the endothelial barrier (893.7)

Abstract

VE‐cadherin, the major endothelial adhesion molecule of AJs, forms hemophilic adhesion and induces re‐organization of actin and microtubule (MT) cytoskeleton. In the current study, we have addressed the role of MT end binding (EB) protein 3 in regulating stability of VE‐cadherin adhesion and lung tissue‐fluid homeostasis. Depletion of EB3 significantly attenuated inositol 1,4,5‐thriphosphate (IP3)‐gated release of calcium from intracellular stores and cell shape change in response to serine protease thrombin. Using acceptor‐photobleaching FRET and pulldown experiments we demonstrated that EB3 interacted with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) both in cells and in cell‐free system. Immunofluorescent staining of endogenous IP3R3 and live‐cell imaging of exogenously‐expressed IP3R3‐GFP in EB3 depleted cells showed that the interaction between EB3 and IP3R3 at the MT tip is required for IP3R3 clustering. Rescue with EB3 mutant lacking the last four amino acids did not restored IP3R3 clustering suggesting that presence of acidic tail is critical for interaction with IP3R3. Pre‐treatment of cells with membrane permeant variant of IP3R3‐derivative peptide (IPRP), containing the EBs consensus motif, significantly inhibited the interaction between EB3 and IP3R3 as well as attenuated thrombin‐induced release of calcium from intracellular stores. Furthermore, infusion of IPRP in lung microvasculature significantly inhibited increase in lung vascular liquid permeability (Kf,c) in response to activation of Protease‐activated receptors‐1 with an agonist peptide. These results identify a novel function of EB3 in regulating calcium signaling and tissue‐fluid homeostasis in lung.