Epithelial-specific isoforms of protein 4.1R promote adherens junction assembly in maturing epithelia

Shu-Ching Huang,Jia Y Liang,Long V Vu,Faye H Yu,Alexander C Ou,Jennie Park Ou,Henry S Zhang,Kimberly M Burnett,Edward J Benz

doi:10.1074/jbc.ra119.009650

Abstract

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of β-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.

Highlights

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states
Given the known role of 4.1R exon 16 in regulating the spectrinbased plasma membrane skeleton [34, 36] and our findings that exon 17b– encoded peptides interact with actin, it is reasonable to postulate that expression of 4.1R forms lacking both exons 16 and 17b may have a negative effect on the organization of the membrane-associated cytoskeleton
Interplay between the peri-junctional F-actin cytoskeleton and the plasma membrane is essential for the integrity and remodeling of epithelial AJs during normal epithelial morphogenesis

Summary

To whom correspondence should be addressed

Dept. of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215. Studies of the reorganization of the cadherin– catenin complex in AJs have identified actin-polymerizing, bundling, and motor proteins associated with AJs. For example, formin-1 is an actin nucleator that binds ␣-catenin and localizes to AJs [19], and cortactin is recruited to cell– cell adhesive contacts and binds to p120-catenin upon homophilic cadherin interaction [20]. Junctional recruitment and stabilization of ␤II-spectrin at the cell– cell contact are critical for the formation of AJs in human epithelia [26]. It mediates the recruitment of spectrin to actin filaments and co-localizes with E-cadherin/␤-catenin at AJs. The spectrin–adducin-based membrane skeleton serves as an important regulator of AJ integrity and remodeling [28]. Its disruption impairs formation of the highly-ordered spectrin lattice at the plasma membrane of contacting cells and results in attenuated junctional assembly. Our results offer new insights into the biological function of 4.1Rϩ17b forms in stabilization of E-cadherin– dependent adhesion and suggest a molecular mechanism by which 4.1R modulates AJ assembly during junctional remodeling

Results

Discussion

Experimental procedures