Dissection of αβ integrin regulation by Rap1 using novel conformation-specific monoclonal anti-β antibodies.

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in αβ integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn-dependent epitopes of β. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of αβ. The induction of colitis by Rap1-deficient CD4 effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on αβ is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).