Epitope mapping of antibodies to the C-terminal region of the integrin β₂ subunit reveals regions that become exposed upon receptor activation

The cysteine-rich repeats in the stalk region of integrin β subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the β₂ subunit, one, KIM127, preferentially bound to α_Lβ₂ that was activated by mutations in the cytoplasmic domains, and by Mn²⁺. KIM127 also bound preferentially to the free β₂ subunit compared with resting α_Lβ₂ Activating β₂ mutations also greatly enhanced binding of KIM127 to integrins α_Mβ₂ and α_Xβ₂. Thus, the KIM127 epitope is shielded by the α subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated α_Lβ₂ and bound equally well to resting and activated α_Lβ₂, differentially recognized resting α_Mβ₂ and α_Xβ₂, and bound fully to activated α_Mβ₂ andα_Xβ₂. The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define rive different β₂ stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.