Dissection of α(4)β(7) integrin regulation by Rap1 using novel conformation-specific monoclonal anti-β(7) antibodies

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α(4)β(7) integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β(7) chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn(2+)-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(2+)-dependent epitopes of β(7). 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(7) was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β(7) was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α(4)β(7). The induction of colitis by Rap1-deficient CD4(+) effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α(4)β(7) is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).