Abstract
Purpose :
Previous studies have demonstrated that norrin restores blood-retinal barrier (BRB) properties after vascular endothelial growth factor that requires both a β-catenin dependent pathway and a non-canonical pathway that involves the direct interaction between disheveled-1 (DVL1) and claudin-5 (CLDN5). Here, we explore the mechanism by which norrin promotes the binding of DVL1 and CLDN5 to induce BRB restoration. Recent evidence indicates that the DVL3 PDZ-binding motif at the C-terminus can loop back and bind to the PDZ domain of DVL, forming a closed conformation. Moreover, the phosphorylation of the PDZ domain by CKe releases the C-terminus and promotes an open DVL conformation. Since DVL1 interacts with CLDN5 through its PDZ domain, we hypothesize that norrin induced phosphorylation promotes a DVL1 conformational change that favors the exposure of the DVL1 PDZ and promotes interaction with CLDN5.
Methods :
In primary bovine retinal endothelial cells (BREC), the phosphorylation of DVL induced by norrin was analyzed by gel shift on Western blot and after alkaline phosphatase. The interaction between the HA-DVL1 variant mimicking phosphorylation (S268E/S311E) and the non-phosphorylatable variant (S268A/S311A) with CLDN5 was determined by co-immunoprecipitation in HEK293 cells. We evaluated the closed or open conformation of DVL1 induced by CK1e by FLIM-FRET.
Results :
On cell lysates from BREC monolayers stimulated with norrin, VEGF, or both for 72 hours, the Pan-DVL antibody detected at least two bands that are likely forms of DVL phosphorylation. The alkaline phosphatase treatment collapsed these bands into one with a lower molecular weight. The co-immunoprecipitation assays demonstrated that the interaction of HA-DVL1 S268E/S311E with CLDN5 is stronger than with HA-DVL1-WT, and the HA-DVL1 S268A/S311A is less stable and interacts less with CLDN5. Finally, in HEK293 cells, the FRET efficiency of the ECFP-DVL1 sensor was higher with CKe 1inhibition.
Conclusions :
These results demonstrate that norrin promotes DVL phosphorylation and that the DVL1-PDZ phosphorylation sites (S268 and S313) promote an open conformation and regulate the interaction with CLDN5. These data reveal a novel signaling pathway involving DVL1 phosphorylation and CLDN5 interaction that is required by norrin to promote barrier regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.