Highly conserved talin isoforms encode distinct mechanical responses and ligand-binding capabilities

Talin is a central mechanosensitive adaptor that links integrins to the actin cytoskeleton and converts mechanical force into biochemical signals. Vertebrates express two highly homologous talin isoforms, talin-1 and talin-2, yet the molecular basis for their nonredundant functions remains poorly understood. Here, we investigate how isoform-specific sequence differences alter the mechanical response and ligand-binding capabilities of the talin rod. Using single-molecule force spectroscopy and biochemical binding assays, we show that talin-2 preserves the overall mechanical architecture of talin-1 but exhibits distinct mechanical responses in key rod regions. Talin-2 R7-R9 exhibits a previously unobserved three-domain cooperative unfolding pathway, while an alternative sequential pathway reveals early unfolding of R9 at substantially lower force than in talin-1. Strikingly, talin-2 R4-R6 has acquired two additional mechanically regulated interactions: a previously unrecognized vinculin-binding site in R5 and an A-kinase anchoring protein (AKAP) region within R5-R6 that recruits protein kinase A (PKA). Together with the conserved R9 AKAP site, this establishes two spatially separated, mechanically regulated PKA-anchoring regions within the talin-2 scaffold. These findings establish that isoform-specific sequence divergence not only reshapes the mechanical response of the talin scaffold but also expands its repertoire of force-regulated interactions, providing a mechanism for the assembly of different structural and signaling complexes in an isoform-dependent manner.