Molecular basis for differential PIP2-mediated association between Vinculin and its splice isoform Metavinculin.

Abstract

Vinculin (Vcn) and its splice variant metavinculin (MVcn) are cell adhesion proteins that regulate cell morphology, adhesion and motility. They function as scaffold proteins that anchor membrane receptors to filamentous actin (F-actin) at focal adhesions (FA) and cell-cell junctions. MVcn bears an extra 68 amino acid insert in the tail domain and is selectively expressed in cardiac and smooth muscle cells at sub-stoichiometric levels relative to Vcn. Mutations in the MVcn tail domain (MVt) promote cardiomyopathy, yet how these mutations alter ligand interactions to promote defects in force transduction and reduced blood flow is unclear. One difference between Vcn and MVcn lies in the ability to reorganize F-actin, with MVcn negatively regulating Vcn-mediated F-actin bundling. Vcn associates with phosphatidylinositol 4,5-bisphosphate (PIP2) through its tail domain (Vt) to drive recruitment, activation and FA turnover. However, it remains unclear whether MVcn specifically associates with PIP2-containing membranes and how such interactions might influence its functional interplay with Vcn in tissues where both isoforms coexist. To evaluate the interaction of MVt and MVt cardiomyopathy mutants with PIP2-membranes in comparison with Vt, we conducted mutagenesis, phospholipid-association assays and computational modeling. We found that MVt shows reduced association for PIP2-containing liposomes relative to Vt due to sequence differences within the insert region. Moreover, mutations in MVt that promote cardiomyopathies do not affect PIP2-dependent lipid association. These findings suggest that MVcn differs from Vcn in driving PIP2-mediated membrane association and sheds light on the coordinate role of Vcn and MVcn in membrane association as well as MVcn cardiomyopathy defects.