PT  - JOURNAL ARTICLE
AU  - Jonathon A. Heier
AU  - Sabine Pokutta
AU  - Ian W. Dale
AU  - Sun Kyung Kim
AU  - Andrew P. Hinck
AU  - William I. Weis
AU  - Adam V. Kwiatkowski
TI  - Distinct autoinhibitory mechanisms regulate vinculin binding by αT-catenin and αE-catenin
AID  - 10.1101/2020.10.25.354415
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.10.25.354415
4099  - http://biorxiv.org/content/early/2020/10/26/2020.10.25.354415.short
4100  - http://biorxiv.org/content/early/2020/10/26/2020.10.25.354415.full
AB  - α-catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of αT(testes)-catenin, an α-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that αT-catenin binds the β-catenin/N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.