JAM‐A –mediated regulation of epithelial barrier function is dependent on a complex with ZO‐2, Afadin and PDZ‐GEF1 that modulates Rap2c activity.

Abstract

Intestinal mucosal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional Adhesion Molecule A (JAM‐A) is a TJ‐associated protein known to regulate epithelial barrier, however mechanisms linking JAM‐A to regulation of permeability are incompletely understood. Proteomic, biochemical and cell‐biological approaches were used to identify candidate proteins involved in JAM‐A regulation of epithelial permeability. Our findings suggest that JAM‐A directly binds to the scaffold molecule ZO‐2 and indirectly associates with Afadin and PDZ‐GEF1 to regulate activation of the small GTPase Rap2c. A link to regulation of barrier was confirmed by siRNA‐mediated downregulation of these regulatory proteins leading to enhanced permeability. Since ZO‐2, Afadin and Rap2c have been implicated in cytoskeletal dynamics, we investigated whether JAM‐A regulation of barrier function is mediated through peri‐junctional actomyosin. While photobleaching experiments suggest that JAM‐A does not regulate actin turnover, JAM‐A deficient cells display enhanced activation of RhoA, a known inducer of actomyosin contraction. These findings suggest that JAM‐A regulates epithelial permeability through association with a multimolecular signaling complex that activates Rap2c, modulating contraction of the peri‐junctional actin cytoskeleton.