JAM‐A regulates epithelial integrin expression and cell shape by activation of Rap1a and Rap1b through a complex with Afadin and PDZ‐GEFs

Abstract

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein implicated in the regulation of epithelial β1 integrin expression, cell shape and barrier function. We previously determined that this regulation requires both dimerization and PDZ binding motifs on different regions of JAM‐A. To gain further insight into JAM‐A mediated signaling in vitro, we downregulated expression of scaffolding proteins and found that loss of Afadin resulted in cellular changes similar to those observed with loss of JAM‐A. Immunofluorescence (IF) and immunoprecipitation (IP) studies revealed co‐association of Afadin with JAM‐A. Interestingly, the small GTPase Rap1 is known to interact with Afadin, regulate integrin function and have decreased activity after loss of JAM‐A. Downregulation of Rap1a resulted in decreased β1 integrin protein levels whereas loss of Rap1b resulted in altered cell shape. Since Rap1 activation is dependent on guanine nucleotide exchange factors (GEFs) we assessed expression of GEFs in vitro and identified PDZ‐GEF1 and PDZ‐GEF2 as major transcripts in colonic epithelia. IF and IP studies revealed co‐association of the PDZ‐GEFs with JAM‐A and loss of the PDZ‐GEFs resulted in diminished Rap1 activity. These results suggest that dimerized JAM‐A signals through a complex that may contain both Afadin and PDZ‐GEFs which, in turn, activate Rap1a and Rap1b to regulate β1 integrin levels and cell shape.Supported by NIH DK‐61379