Load-dependent RGD-context sensing via αV-class integrins reprograms cell adhesion and mechanics

The biochemical and mechanical properties of extracellular matrix proteins govern cell adhesion, mechanics, and migration. How cells use integrins to discriminate between the arginine-glycine-aspartic acid motifs presented by different extracellular matrix proteins, a process central to tissue homeostasis and disease, has remained unclear. Here we show that mammalian cells mount a distinct “biphasic” mechanical response through αV-class integrins to the arginine-glycine-aspartic acid motif of vitronectin compared with fibronectin, osteopontin, and cyclic arginine-glycine-aspartic acid. Within seconds of contact with vitronectin, we find that αV-class integrins strengthen cell adhesion through two load-dependent mechanotransduction pathways in which αVβ3 and αVβ5 integrins take complementary roles. Under low load, we demonstrate that the first phase requires both integrins together with an intact, pre-tensed actomyosin cortex, talin, paxillin, and focal adhesion kinase activity, with αVβ5 integrin additionally engaging clathrin-mediated endocytosis. Under higher load, we show that the second phase is dominated by αVβ3 integrin–directed actin-related protein 2/3, cellular Src kinase, and phosphatidyl inositol-3-kinase signaling, which organizes the consensus adhesome, while αVβ5 integrin concurrently drives cellular stiffening. Taken together, we find that αV-class integrins rapidly deploy arginine-glycine-aspartic acid -motif- and β-subunit-specific programs that cooperatively tune cell adhesion and mechanics according to the extracellular matrix composition.