The EC2 domains of tetraspanins CD9, CD81, and CD151 bind to the allosteric site of integrins (site 2) and activate integrins αvβ3, α5β1 and α4β1 in a biphasic manner.

Previous studies showed that tetraspanins activate integrins, but the mechanism of this action is unclear. We previously showed that the extracellular-2 (EC2) domains of CD9, CD81, and CD151 bind to the classical RGD-binding site (site 1) of integrin v{beta}3, suggesting that they are integrin ligands. We showed that several inflammatory cytokines (e.g., CX3CL1, CXCL12, CCL5, and CD40L) bind to the allosteric site (site 2) of integrins, which is distinct from site 1, and activate integrins (allosteric activation). 25-hydroxycholesterol, a major inflammatory lipid mediator, is known to bind to site 2 and induce inflammatory signals, suggesting that site 2 plays a role in inflammatory signaling. We hypothesized that the EC2 domains activate integrins by binding to site 2. Here we describe that docking simulation predicted that CD81 EC2 binds to site 2 of v{beta}3 and more strongly to site 2 of 5{beta}1. Peptide from site 2 bound to isolated EC2 domains, suggesting that the EC2 domains bind to site 2. The EC2 domains only weakly activated v{beta}3 but more efficiently activated cell surface integrins 5{beta}1 and 4{beta}1 on the cell surface. These results are consistent with the previous findings that these tetraspanins preferentially interact with {beta}1 integrins. The integrin activation by the EC2 domains was increased at low EC2 concentrations and reduced as EC2 concentrations increased (biphasic), which is consistent with the findings that the EC2 domains bind to two sites (site 1 and 2). We propose that the EC2 binding to site 2 is a novel target for drug discovery.