In order to identify a binding site for ligand intercellular adhesion molecule‐1 (ICAM‐1) on the beta 2 integrin lymphocyte function‐associated antigen‐1 (LFA‐1), protein fragments of LFA‐1 were made by in vitro translation of a series of constructs which featured domain‐sized deletions starting from the N‐terminus of the alpha subunit of LFA‐1. Monoclonal antibodies and ICAM‐1 were tested for their ability to bind to these protein fragments. Results show that the putative divalent cation binding domains V and VI contain an ICAM‐1 binding site. A series of consecutive peptides covering these domains indicated two discontinuous areas as specific contact sites: residues 458‐467 in domain V and residues 497‐516 in domain VI. A three‐dimensional model of these domains of LFA‐1 was constructed based on the sequence similarity to known EF hands. The two regions critical for the interaction of LFA‐1 with ICAM‐1 lie adjacent to each other, the first next to the non‐functional EF hand in domain V and the second coinciding with the potential divalent cation binding loop in domain VI. The binding of ICAM‐1 with the domain V and VI region in solution was not sensitive to divalent cation chelation. In short, a critical motif for ICAM‐1 binding to the alpha subunit of LFA‐1 is shared between two regions of domains V and VI.

The integrin receptors on leukocytes are transiently activated by "triggering" molecules that may be other leukocyte membrane structures such as the T-cell receptor complex or small molecules such as PAF, which bind to their own specific receptors. This "inside out" signaling is essential for high affinity integrin/ligand pairing. In the example of LFA-1/ICAM-1, binding is positively supported by Mg2+ but negatively supported by Ca2+. How specific divalent cations affect receptor activation and subsequent ligand binding has still to be fully understood. However, the fact that activation can be mimicked from outside the cell via special anti-LFA-1 monoclonal antibodies such as MEM-83 suggests that activated integrins undergo conformational changes. Further alteration occurs as a result of the interaction of integrin with ligand, and the resulting novel epitopes are named "ligand-induced binding sites." For a brief period of time the integrin/ligand complex is able to transmit signals from "outside in." The transient activation of leukocyte integrins determines that cell-cell adhesion will be short lived and serves the purpose of permitting recycling of effector cells with their targets.

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Cultured T cells and freshly isolated mononuclear leukocytes are able to bind collagen specifically. These leukocytes express equivalent levels of the integrins VLA-1, VLA-2 and VLA-3 which are collagen-binding receptors on other cells. However, only solubilized VLA-2 is able to bind collagen and only monoclonal antibodies specific for alpha 2 or beta 1 subunits are able to block the binding of intact cells to collagen. This restriction provides another example of the dependence of integrin specificity on the cell type on which it is expressed. It was also speculated that the inserted or I domain on the alpha subunits of VLA-1, VLA-2 and the beta 2 integrin family might have a role in collagen binding on the basis of its sequence homology to other types of collagen binding proteins. However, LFA-1, CR3 and p150,95 showed no collagen binding activity, suggesting that the I domain has another function.

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