Superactivation of integrin αvβ3 by low antagonist concentrations

Abstract

Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin (α)v(β)3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant (α)v(β)3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rs(α)v(β)3and native purified (α)v(β)3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of sr(α)v(β)3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rs(α)v(β)3 and native purified (α)v(β)3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin (α)v(β)3 to a comparable level as activation by manganese.

Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations

“…54,57,58 The binding of RGD-based antagonists to αVβ3 produces similar changes in metal-ion coordination to those observed with the RGD-based antagonists to αIIbβ3 59 and also exposes a ligand-induced binding site (LIBS) on the β3 PSI domain for mAb AP5. 60−62 This partial agonist activity may contribute to inducing receptor extension, 63 priming the receptor to bind ligand at low doses in vitro, 64 enhancing angiogenesis ex vivo at sub-IC 50 concentrations, 62 and activating osteoclast signaling like that produced by natural ligands. 65 Many of the animal studies supporting a role for αVβ3 in the pathophysiology of disease have included evidence that cilengitide improves outcome.…”

High-Affinity RGD-Knottin Peptide as a New Tool for Rapid Evaluation of the Binding Strength of Unlabeled RGD-Peptides to αvβ3, αvβ5, and α5β1 Integrin Receptors

“…To better analyze the effect of the cations, we only immobilized 0.25 μg/mL of α v β 3 . Even though the effects were marginal, the use of 0.5 mM of Mn 2+ instead of, or in addition to, 0.5 mM Mg 2+ (also tested by Legler et al 28 ) led to slightly lower binding (10 nM of 1a: OD ∼ 0.35 iso. 0.38), while the use of Mg 2+ alone at 1.0 mM even slightly increased the binding of 1a (10 nM of 1a: OD ∼ 0.41 iso.…”

“…Based on a report by Legler et al in 2001 that the affinity of α v β 3 to ligands, such as fibronectin or vitronectin, can be modulated using bivalent cations, 28 we then repeated our binding studies with 1a and α v β 3 in the presence of 1.0 mM CaCl 2 and 0.5 mM MgCl 2 in all buffers. In the presence of these cations, we found that binding of peptide 1a to α v β 3 was drastically improved and could be detected (OD ∼ 1.0) at 0.5 μg/mL of α v β 3 and 0.1 nM of 1a, respectively, which is equivalent to a ∼10000-fold reduction (Figure 2A).…”

Tumor Targeting by Peptide-Decorated Gold Nanoparticles

“…The inhibition of network formation at a lower concentration can be reliably ascribed to the ability of GRGDG-NH 2 to bind and block the αvβ3 integrin by acting as an integrin antagonist blocking or decreasing cell migration and thus network formation . Indeed, RGD-like peptides are able to block the integrin binding site by competing with the RGD-containing ligands (vibronectin, fibronectin) . Network destruction by RGD-AuNPs and GRGDG-NH 2 could be due to the ability of small RGD-like peptides to act as potent integrin antagonist compounds that can disrupt the αvβ3 integrin–ligand interactions …”

“…75 Indeed, RGD-like peptides are able to block the integrin binding site by competing with the RGDcontaining ligands (vibronectin, fibronectin). 76 Network destruction by RGD-AuNPs and GRGDG-NH 2 could be due to the ability of small RGD-like peptides to act as potent integrin antagonist compounds that can disrupt the αvβ3 integrin−ligand interactions. 77 Overall, the results obtained in the in vitro experiments can be explained by considering (i) the overexpression of αvβ3 integrin in the B16F10 cell line, (ii) the affinity of RGD-like peptides for αvβ3 integrin, and (iii) the critical role of this integrin isoform in cell migration and tumor growth.…”