Cytoplasmic salt bridge formation in integrin αvß3 stabilizes its inactive state affecting integrin-mediated cell biological effects.

Heterodimeric integrin receptors are mediators of cell adhesion, motility, invasion, proliferation, and survival. By this, they are crucially involved in (tumor) cell biological behavior. Integrins trigger signals bidirectionally across cell membranes: by outside-in, following binding of protein ligands of the extracellular matrix, and by inside-out, where proteins are recruited to ß-integrin cytoplasmic tails resulting in conformational changes leading to increased integrin binding affinity and integrin activation. Computational modeling and experimental/mutational approaches imply that associations of integrin transmembrane domains stabilize the low-affinity integrin state. Moreover, a cytoplasmic interchain salt bridge is discussed to contribute to a tight clasp of the α/ß-membrane-proximal regions; however, its existence and physiological relevance for integrin activation are still a controversial issue. In order to further elucidate the functional role of salt bridge formation, we designed mutants of the tumor biologically relevant integrin αvß3 by mutually exchanging the salt bridge forming amino acid residues on each chain (αvR995D and ß3D723R). Following transfection of human ovarian cancer cells with different combinations of wild type and mutated integrin chains, we showed that loss of salt bridge formation strengthened αvß3-mediated adhesion to vitronectin, provoked recruitment of cytoskeletal proteins, such as talin, and induced integrin signaling, ultimately resulting in enhanced cell migration, proliferation, and activation of integrin-related signaling molecules. These data support the notion of a functional relevance of integrin cytoplasmic salt bridge disruption during integrin activation.

The glycophorin A transmembrane sequence within integrin αvß3 creates a non-signaling integrin with low basal affinity that is strongly adhesive under force.

Integrin heterodimeric cell adhesion and signaling receptors bind ligands of the extracellular matrix and relay signals bidirectionally across cell membranes. Thereby, integrins adopt multiple conformational and functional states that control ligand binding affinity and linkage to cytosolic/cytoskeletal proteins. Here, we designed an integrin chimera encompassing the strongly dimerizing transmembrane domain (TMD) of glycophorin A (GpA) in the context of the otherwise unaltered integrin αvß3. We hypothesized that this chimera should have a low basal affinity to soluble ligand but should be force-activatable. By cellular expression of this chimera, we found a decreased integrin affinity to a soluble peptide ligand and inhibited intracellular signaling. However, under external forces applied by an atomic force microscope or by a spinning disc device causing shear forces, the mutant caused stronger cell adhesion than the wild-type integrin. Our results demonstrate that the signaling- and migration-incapable integrin αvß3-TMD mutant TMD-GpA shows the characteristics of a primed integrin state, which is of low basal affinity in the absence of forces, but may form strong bonds in the presence of forces. Thus, TMD-GpA may mimic a force-activatable signaling intermediate.