Abstract
Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.
Highlights
Integrins are a family of a/b heterodimeric transmembrane cell surface receptors that mediate the cell-extracellular matrix and cell-cell interactions
All nine anti-aVb3 monoclonal antibodies (mAbs) that were tested bound to cells expressing wild-type aVb3 but not to cells expressing aIIbb3 or to parent Chinese hamster ovary (CHO) cells, with the exception of 10C4, 23C6, and LM609, which showed a partial reactivity with cells expressing aIIbb3 (Table 1)
17E6 and 69-6-5 bound to cells expressing V/B, but not to cells expressing B/V (Table 1). These results clearly indicated that the epitopes for mAbs 17E6 and 69-6-5 are contained in the N-terminal bpropeller domain and that the epitopes for mAbs AMF-7, M9, P2W7, and P3G8 are contained in the C-terminal leg region, consisting of the thigh, calf-1, and calf-2 domains
Summary
Integrins are a family of a/b heterodimeric transmembrane cell surface receptors that mediate the cell-extracellular matrix and cell-cell interactions. The head region points downward, facing the plasma membrane The discrepancies between these two structures were reconciled by a high-resolution EM image of the extracellular domains of recombinant aVb3 integrin [5]. The transition from one conformer to the other (the so-called switchblade-like movement) might account for the affinity regulation of the integrin Consistent with these findings, genetically engineered aIIbb constrained in the bent state interfered with the binding of macromolecular ligands, while aIIbb constrained in the extended state exhibited maximal activation [6,7]. The crystal structures of aIIbb head regions complexed with short ligand peptides or ligand mimetics have provided detailed information [3,10] This swing-out movement is accompanied by the rearrangement of the ligand-binding and/or cation-binding loops in the bA domain, thereby regulating ligand binding. These results suggest that extension and an open headpiece conformation are independently required for high-affinity ligand binding
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