Abstract
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. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry 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 the 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 show 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.
Highlights
The cadherin–catenin complex that forms the core of the adherens junction (AJ) is required for intercellular adhesion and tissue integrity [1,2,3]
The adhesive properties of classical cadherins are driven by the recruitment of cytosolic catenin proteins to the cadherin tail: p120-catenin binds to the juxtamembrane domain
ΑT-catenin forms a strong cadherin–catenin core complex like αE-catenin to link actin to the AJ. αT-catenin is coexpressed with αEcatenin in multiple mammalian tissues and, in the heart, it is enriched along the intercalated disc (ICD) with αE-catenin [39, 45, 46, 50]
Summary
The cadherin–catenin complex that forms the core of the adherens junction (AJ) is required for intercellular adhesion and tissue integrity [1,2,3]. We show that the M-region of αT-catenin is not autoinhibited and can bind the vinculin N-terminus in the absence of tension with strong affinity. We used the head region (comprising the N- and M-domains) of αT-catenin (aa 1–659, Fig. 1A) for these experiments because it is more stable than full-length αT-catenin and yields sufficiently high protein concentrations for ITC.
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