KRIT1 stabilizes endothelial adherens junctions independent of Rap1 via regulation of β1‐integrin

• Published in: The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.04041
• Main Authors: Swamy, Harsha, Glading, Angela
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2021.35.S1.04041

The junctional scaffold KRIT1 interacts with adherens junctions (AJ) proteins to stabilize endothelial cell‐cell contacts. Loss of KRIT1 function leads to Cerebral Cavernous Malformations (CCM), a disease characterized by leaky vascular lesions as the result of perturbed endothelial proliferation and inflammatory signaling. As a true Rap1 effector, KRIT1 localizes to and associates with AJ after binding active Rap1. Inhibition of this interaction by mutation of the Rap1‐binding site (KRIT1‐R452E) prevents KRIT1 from localizing to AJ and stabilizing barrier function. However, it remains unclear whether KRIT1 can stabilize endothelial junctions independently of Rap1. In order to study the mechanisms of KRIT1‐mediated junction stabilization, we targeted mCherry‐tagged KRIT1‐R452E to the plasma membrane using a C‐terminal lipid modification (MT‐R452E). Immunofluorescence studies indicated that this construct localized to cell‐cell contacts similar to WT‐KRIT1 in endothelial cells depleted of endogenous KRIT1 (KRIT1 shRNA). While MT‐R452E partially rescued AJ staining in these cells, it was unable to rescue barrier function, suggesting that both KRIT1 and Rap1 may be necessary at AJ to stabilize the endothelial barrier. As Rap1 binding may also regulate a putative intramolecular interaction between KRIT1's first NPxY motif and its C‐terminal PTB domain, we sought to evaluate whether stabilization of endothelial junctions was dependent on KRIT1 conformation. The NPxY motif of MT‐R452E was mutated (N192A‐Y195A) to yield a constitutively open, membrane‐targeted construct (MT‐R452E‐ΔNPxY). Immunofluorescence confirmed that this construct localized to cell‐cell contacts, and appeared to rescue AJ staining after loss of endogenous KRIT1. Interestingly, open conformation R452E lacking any targeting sequence (R452E‐ΔNPxY) also rescued AJ staining despite remaining primarily cytosolic. Further, both open conformation constructs rescued barrier function independent of Rap1 binding, indicating that KRIT1's junction stabilizing effects may be exerted without binding of Rap1 or localization to cell‐cell contacts. As this NPxY (192‐195) motif mediates binding to ICAP1a, which has been shown to regulate β1‐integrin activity, we next examined whether junctional stability was associated with changes in β1‐integrin activation. Using an activation‐specific antibody against β1‐integrin, we observed an increase in large fibrillar adhesions in cells depleted of KRIT1 (KRIT1 shRNA),