Transmembrane Signaling and Conformational Regulation of Integrin

Integrins are heterodimeric cell adhesion molecules that play an essential role in hemostasis, thrombosis, cell migration, and can transmit bidirectional signals. The inside-out activating signal triggers integrin conformational change, leading to high affinity for extracellular ligands. Binding of extracellular matrix ligands to integrins results in outside-in signaling that leads to formation of focal adhesion (FA) complex at the integrin cytoplasmic tails and activation of downstream signal pathways. Transmembrane/cytoplasmic domains are hypothesized to associate in resting integrins, whereas ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the á subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin áIIbâ3 constructs, we discovered that an áIIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In addition, preventing transmembrane domain separation abolished both inside-out and outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. It has been proposed that integrins adopt a bent, low affinity conformation under physiological conditions and can switch to an extended conformation through a “switchblade”-like conformational change. It is extremely important to verify if the model universally applies to all integrin families. Due to the highly divergent cytoplasmic domain, â8 integrin is unlikely to adopt a general mechanism for affinity regulation. Therefore the â8 integrin may uniquely assume an extended, high affinity conformation under physiological conditions. We discovered that â8 indeed showed high binding to vitronectin under physiological conditions. Further studies identified that the I-EGF domains on the â8 integrin extracellular lower leg are critical for this high affinity conformation. We also pinpointed a critical role of the â8 integrin EGF domain 1 and 2 in this high affinity conformation. Mutating either one of the cysteine pair at the â3 EGF1 domain C-terminus resulted in high affinity conformation, probably due to disruption of the EGF1-EGF2 interface. Our study also showed that the â8 ectodomain lower stalk does not negatively affect outside-in signaling