Abstract
The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.
Highlights
MAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the ␣ and  subunit knees to outside to in)
We show that: (i) monoclonal antibodies N29 and 8E3 have epitopes in the 1 subunit PSI domain and stimulate ligand binding to ␣51; (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the ␣ subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the ␣ subunit
Both mAbs have epitopes at the extreme N terminus of the PSI domain, and we show that binding of these antibodies to the integrin is likely to wedge apart the ␣ and  knee regions, resulting in unbending
Summary
Monoclonal Antibodies—Mouse anti-human 1 mAbs 12G10 and 8E3 were produced as described previously [22, 23]. To create a construct containing only the thigh and calf-1 domains (C1␣5) the truncation position between calf-1 and calf-2 domains (amino acid Ala749) was chosen based on alignment of the ␣5 subunit sequence with the ␣V subunit structure [6] Both ␣ and  constructs were fused in-frame to the hinge regions and CH2 and CH3 domains of human IgG␥1 (i.e. the Fc portion of the heavy chain). The blocking solution was removed, and the wells were washed three times with 200 l of 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml bovine serum albumin (buffer A). Peroxidase-conjugated anti-mouse Fc secondary antibody (1:1,000 dilution in buffer A; Jackson Immunochemicals) was added (50 l/well) for 30 min, the plate washed four times in buffer A, and color was developed using ABTS substrate. Each experiment shown is representative of at least three separate experiments
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