Evidence That Monoclonal Antibodies Directed against the Integrin β Subunit Plexin/Semaphorin/Integrin Domain Stimulate Function by Inducing Receptor Extension

Abstract

The overall structure of integrins is that of a ligandbinding 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 ␣ and ␤ subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the ␤ subunit. The role played by the knee domains in the regulation of integrinligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the ␤ 1 subunit PSI domain and stimulate ligand binding to ␣ 5 ␤ 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 ␣ 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. 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 ␣ and ␤ 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 ␣ 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.Integrins provide a crucial bridge between the inside and outside environments of the cell by linking the surrounding matrix of a cell to its cytoskeletal framework (1). These receptors are ␣,␤ heterodimers, and both subunits have large extracellular domains and short intracellular regions. Integrins carry a two-way flow of information (inside the cell to out, and outside to in). To achieve this bidirectional signaling integrins must convey shape changes over a long distance, from the intracellular domains to the extracellular regions and vice versa (2, 3). Furthermore, in most cases binding of integrins to their extracellular ligands has to be tightly controlled. For example, the interaction of ␣ IIb ␤ 3 with fibrinogen during platelet aggregation needs to be restricted to sites of vessel injury. Regulation of ligand binding is achieved by switching of an integrin between a constitutive low affinity (inactive) state and a high affinity (primed) state. In addition, the interaction of ligands with integrin stabilizes the high affinity state and may cause further shape shifting (ligand-activated state) (4, 5). However, the molecular basis of the conformational changes involved is currently uncertain.The recent crystal structures of the extracellular domains of ␣ V ␤ 3 (6, 7) have provided new insights into integrin function. Overall, the integrin structure resembles that of a "head" on two "legs."...

Structural Basis of Integrin Regulation and Signaling

“…The bends in the α leg at the genu and in the β leg between I-EGF1 and I-EGF2 are located close to one another and in a geometry appropriate so that extension can occur by pivoting of the headpiece about an axis through the α and β subunit knees ( Figure 6c,d), as shown by EM studies (33,43). Consistent with these findings, many antibodies that either activate or report activation in cell surface integrins map to the PSI and β I-EGF domains (44)(45)(46)(47). Furthermore, αL antibodies that report extension map to the inner face of the thigh domain and require genu Ca 2+ -coordinating residues for binding and thereby provide evidence that integrin extension occurs by a rearrangement at the thigh/genu interface (48).…”

Expression of Plxdc2/TEM7R in the developing nervous system of the mouse

“…This gene encodes a type I transmembrane protein of 530 amino acids, characterised by an extracellular region of weak nidogen homology and a plexin repeat or PSI domain, a domain found in known axon guidance molecules such as semaphorins and plexins as well as integrins and a small number of other molecules (Bork et al, 1999). The PSI domain has been shown to be involved in autoregulatory intramolecular contacts in a number of cases (Bunch et al, 2006;Mould et al, 2005;Zang and Springer, 2001). A related gene (now called Plxdc1) was isolated in a screen for genes upregulated in human tumour endothelium and named tumour endothelial marker 7 (TEM7) (Carson-Walter et al, 2001;St Croix et al, 2000).…”

Up-Regulation and Activation of Eosinophil Integrins in Blood and Airway after Segmental Lung Antigen Challenge

“…On circulating eosinophils of dual responders the expression level of the epitope for activation-sensitive anti-␤ 1 mAb N29 (34,40,41) was higher 48 h after Ag segmental challenge as compared with the level before challenge (Fig. 2B).…”