Junctional Adhesion Molecule A (JAM‐A) signals through Afadin and Rap1

Abstract

JAM-A is a transmembrane tight junction protein involved in regulation of barrier function and leukocyte transmigration, yet the mechanisms governing these functions are incompletely understood. The extracellular domain of JAM-A is known to participate in protein-protein interactions while the cytoplasmic tail of JAM-A has a PDZ motif that interacts with scaffolding proteins such as Afadin and ZO-1 that are linked to signaling molecules such as small GTPases. We have previously shown that JAM-A siRNA-mediated knockdown causes a decrease in the levels of β1 integrin in an epithelial cell line (SKCO15). We now provide evidence that this decrease in β1 integrin is due to loss of Rap1a activity and that loss of β1 integrin is due to aberrant recycling. Furthermore we show that siRNA-mediated knockdown of JAM-A, Afadin, and Rap1b, but not Rap1a, results in dramatic alterations in epithelial cell shape characterized by loss of cell-cell interactions, flattening and abundant spikes. Such alterations in cell shape appeared to be dependent upon dimerization of JAM-A. Additionally, decreases in JAM-A, Rap1b, and Afadin expression were observed to be associated with an increase in expression of the actin monomer shuttle, Twinfillin. Knockdown of both Twinfillin and JAM-A resulted in partial reversal of the morphologic alterations observed with knockdown of JAM-A alone. Together, these findings suggest that JAM-A dimerization promotes activation of Rap1, presumably mediated by Afadin. We also predict that Rap1b activation is associated with increased twinfillin expression, which alters actin cytoskeleton dynamics and cell morphology. Supported by NIH R01 DK61379