Junctional adhesion molecule-A (JAM-A) is a transmembrane
tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal
immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/
zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of
beta1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules
Afadin and PDZ-
guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both
Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with
Afadin was dependent on the expression of JAM-A. Loss of JAM-A,
Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1,
beta1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased
beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with
Afadin and PDZ-GEF2 that activates Rap1A, which regulates
beta1 integrin levels and cell migration.