An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

Identification and characterization of a human monoclonal antagonistic antibody AL-57 that preferentially binds the high-affinity form of lymphocyte function-associated antigen-1

“…The affinity and kinetics of the locked-open I domain for ICAM-1 are comparable with that determined previously for the activated ␣ L ␤ 2 complex [23], indicating that the ␣ L I domain, when locked in an open conformation, is sufficient for maximal affinity-binding to ICAM-1 [15]. When expressed on the cell surface, the locked-open I domain mediates cell adhesion as potently as maximally activated wild-type ␣ L ␤ 2 [13,14]. The open I domain also can antagonize LFA-1-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion on high endothelial venules [15].…”

“…Stable K562 cell lines that express ␣ L ␤ 2 heterodimers containing locked-open or -closed I domains were established as described previously [13]. Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, puromycin (4 g/ml), penicillin (100 Units/ml), and streptomycin (100 g/ml).…”

“…The ␣ L I domain has been locked in the open or closed conformation with engineered disulfide bonds [11,[13][14][15]. Locking the ␣ L I domain in the open conformation causes a 9000-fold increase in affinity for ICAM-1 compared with the affinity of the inactive wild-type I domain and the locked-closed conformation and can be reversed by disulfide reduction [15].…”

Role of the αI domain in ligand binding by integrin αEβ7

“…Guided by crystal structures, it has been possible to lock the § I domain of § L, § M and § X into an open, active, conformation and to test for ligand binding activity using the complete integrin holoreceptor or the isolated § I domain. This strategy has greatly increased the affinity of the integrin for its ligand and, in the case of § L g 2, has demonstrated that the § I domain provides the sole contact site for ICAM-1 [19][20][21][22][23]. It has been proposed that this picture provides a paradigm for all § I domain-containing integrins [8].…”

Integrin αDβ2, an adhesion receptor up-regulated on macrophage foam cells, exhibits multiligand-binding properties

“…14 The GST component was removed by cleavage with thrombin as described. The "active" ␣ L I-domain, originally described by Lu et al, 19 was prepared using the previously created ␣ L -PGEX4T-1 construct (Gly 128 -Tyr 307 ) 14 as a framework. In the new construct, 2 lysines, Lys 287 and Lys 294 , were substituted to cysteines to create a disulfide bond and, thus, lock the protein in the active conformation.…”