Featured Article - June 2010
Short description: An extended conformation of α5ß1 integrin is important for cell spreading and adhesion complex formation.
Current models for integrin conformation depict the inactive receptor in a bent form, which upon activation becomes fully extended — the legs separate to allow downstream signalling. In the Journal of Cell Biology, Martin Humphries and colleagues now directly put this model to the test for β1 integrin; they determine the conformation of α5β1 integrin in adherent cells and demonstrate that the integrin must be extended for proper formation of focal adhesions.
The authors first tested how the conformation of α5β1 integrin affects its function. By introducing a disulphide bond between the α- and β-subunits of a recombinant, soluble α5β1 integrin fused to an Fc fragment a 'locked-together' (LT) construct was formed, in which the separation of the subunit legs is restricted and the integrin is no longer able to extend.
Binding of an activating anti-β1 monoclonal antibody (mAb) to the 'bending' region of the molecule was inhibited in the LT integrin. Moreover, mutation of various residues identified D522, located in the 'knee' region of the integrin, as the epitope for the anti-β1 mAb. This suggested that restricting leg movement causes the integrin to adopt a bent form.
In addition to leg separation, an outward movement of the hybrid domain is important for ligand-binding affinity. Using stimulatory antibodies against each subunit that stabilize the active conformation of the integrin, the authors showed that the LT integrin displays a lower affinity to ligands, indicating an association between the movements of the legs and the hybrid domain, which together maintain the high affinity receptor.
Next, FRET analysis was used, with specific reporters for the integrin headpiece and the cell membrane, to measure the distance between the two. This approach demonstrated that the ligand-bound α5β1 integrin in adhesion complexes is in an extended conformation.
But, what is the significance of this extended conformation to integrin function? Integrins regulate cell–cell and cell–extracellular matrix (ECM) interactions, which maintain proper adhesion and spreading of cells. Consistently, expression of α5β1 integrin in β1-null cells mediated their spreading and induced formation of adhesion complexes on fibronectin.
Moreover, anti-β1 monoclonal antibody bound to the integrin in these cells, which indicated a high-affinity, activated receptor in its extended form. By contrast, cells expressing the LT integrin formed clusters on the cell surface, but displayed reduced binding to anti-β1 mAb and showed reduced spreading. Consistently, binding of a stimulatory anti-α5 antibody was also inhibited in LT-expressing cells, confirming that prevention of leg separation causes the integrin to adopt an inactive and bent conformation.
Taken together, this study demonstrates that conformational changes are important for the proper functioning of integrins in adherent cells. Specifically, α5β1 integrin needs to be in an extended conformation with free leg movement for the proper spreading of cells and formation of adhesion complexes.
J. A. Askari et al. Focal adhesions are sites of integrin extension.
The Journal of Cell Biology (2010). doi:10.1083/jcb.200907174