New PI(4,5)P₂- and Membrane Proximal Integrin—Binding Motifs in the Talin Head Control β3-Integrin Clustering

Integrin-dependent adhesion sites consist of clustered integrins that transmit mechanical forces and provide signaling required for cell survival and morphogenesis. Despite their importance, the regulation of integrin clustering by the cytoplasmic adapter protein talin (Tal) and phosphatidylinositol (PI)-4,5-biphosphate (PI(4,5)P₂) lipids nor their dynamic coupling to the actin cytoskeleton is fully understood. By using a Tal-dependent integrin clustering assay in intact cells, we identified a PI(4,5)P₂-binding basic ridge spanning across the F2 and F3 domains of the Tal head that regulates integrin clustering. Clustering requires a new PI(4,5)P₂-binding site in F2 and is negatively regulated by autoinhibitory interactions between F3 and the Tal rod (Tal-R). The release of the Tal-R exposes a new β3-integrin—binding site in F3, enabling interaction with a membrane proximal acidic motif, which involves the formation of salt bridges between K³¹⁶ and K³²⁴ with E⁷²⁶ and D⁷²³, respectively. This interaction shields the β-integrin tail from reassociation with its α subunit, thereby maintaining the integrin in a substrate-binding and clustering-competent form.