The Integrin αLβ2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain

Abstract

Integrin activation involves global conformational changes as demonstrated by various functional and structural analyses. The integrin ␤ hybrid domain is proposed to be involved in the propagation of this activation signal. Our previous study showed that the integrin ␤ 2 -specific monoclonal antibody 7E4 abrogates monoclonal antibody KIM185-activated but not Mg 2؉ / EGTA-activated leukocyte function-associated antigen-1 (LFA-1; ␣ L ␤ 2 )-mediated adhesion to ICAM-1. Here we investigated the allosteric inhibitory property of 7E4. By using human/mouse chimeras and substitution mutations, the epitope of 7E4 was mapped to Val 407 , located in the mid-region of the ␤ 2 hybrid domain. Two sets of constitutively active LFA-1 variants were used to examine the effect of 7E4 on LFA-1/ICAM-1 binding. 7E4 attenuated the binding of variants that have modifications to regions membrane proximal with respect to the ␤ 2 hybrid domain. In contrast, the inhibitory effect was minimal on variants with alterations in the ␣ L I-and ␤ 2 I-like domains preceding the hybrid domain. Furthermore, 7E4 abrogated LFA-1/ICAM-1 adhesion of phorbol 12-myristate 13-acetate-treated MOLT-4 cells. Our data demonstrate that interaction between the hybrid and I-like domain is critical for the regulation of LFA-1-mediated adhesion.Integrins are key proteins involved in cell-cell and cell-matrix interactions, mediating essential biological processes such as embryogenesis, the immune response, and inflammation (1, 2). They are heterodimeric type I membrane glycoproteins formed by noncovalent association of an ␣ and a ␤ subunit. In human, 9 of the 18 ␣ subunits have an I (Inserted)-domain found between blades 2 and 3 of a seven-bladed ␤-propeller structure at its N-terminal end. For this particular subset of integrins, the I-domains are involved in ligand binding via their MIDAS 1 motifs (3-6). The ␤ subunit is linearly organized into an N-terminal PSI-domain (for Plexins, Semaphorins, and Integrins) (7), a spacer region, an I-domain like structure (also known as the I-like domain or ␤ A-domain), a mid-region, a cysteine-rich region containing four tandem IEGF (Integrin Epidermal Growth Factor)-domains, and a terminal domain followed by the transmembrane and cytoplasmic segment (Fig. 1A). In the integrins without an I-domain in their ␣ subunits, the ␤ I-like domain participates directly in ligand binding (8).However, in the integrins with an I-domain in their ␣ subunits, the ␤ I-like domain may, in addition, serve a regulatory role in the integrin-mediated adhesion (9). Previously, we constructed integrin ␤ 2 /␤ 7 chimeras in which the N-terminal region (NTR, the combined PSI domain and spacer segment), I-like domain, and the mid-region of the ␤ 2 subunit were replaced with those of the ␤ 7 subunit (10). The epitopes of five ␤ 2 -specific mAbs were mapped by using these chimeras and were found to require both NTR and mid-region for their expression. Coupled with the observation that removal of the I-like domain did not affect the folding of the NTR-midre...

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

“…Interestingly, 7E4 antibody to the hybrid domain has previously been found to inhibit adhesiveness stimulated by agents that act distally to, but not directly on, the βI domain (26). Here, with activation of α X β 2 induced distally by CBR LFA-1/2 Fab, both 7E4 and TS1/18 were found to stabilize the closed headpiece conformation in EM and to abolish adhesiveness to iC3b.…”

“…Similar to the preparation of α X β 2 -CBR LFA-1/2 complex shown in Fig. 2B, approximately equal proportions of particles containing CBR LFA-1/2 Fab and lacking MEM148 Fab adopted closed (14%) and open (16%) headpiece conformations ( mAb 7E4 also binds to the β 2 hybrid domain, but in contrast to MEM148 is inhibitory (25,26). 7E4 binds to residue Val-385, which locates to a loop on the outer face of the hybrid domain, at the interface with the βI domain (Fig.…”

“…Here, with activation of α X β 2 induced distally by CBR LFA-1/2 Fab, both 7E4 and TS1/18 were found to stabilize the closed headpiece conformation in EM and to abolish adhesiveness to iC3b. The ability of Mg 2+ /EGTA and a β 2 N351S mutation, both of which act on the βI domain (26,36), to bypass inhibition by 7E4 (26) suggests that the open conformation of the βI domain, as defined by the conformation of the metal binding sites in the βI domain, can be uncoupled from the open conformation of the headpiece, as defined by hybrid domain swing-out. Such uncoupling has already been observed for β 3 integrins.…”

“…7E4 recognizes Val-385 in a hybrid domain loop adjacent to its interface with the βI domain ( Fig. 1 E and F) (26). Val-385 and the surrounding region are well exposed to solvent in the bent, extended-closed and extended-open integrin conformations (4).…”

“…7E4 inhibits LFA-1 activation on intact cells and can abrogate phorbol ester-or mAb KIM185-activated, β 2 integrin-mediated cell adhesion (26,34). 7E4 recognizes Val-385 in a hybrid domain loop adjacent to its interface with the βI domain ( Fig.…”