Real-time Analysis of Conformation-sensitive Antibody Binding Provides New Insights into Integrin Conformational Regulation

Abstract

Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of ␣ 4 ␤ 1 integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC 50 for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.In the bloodstream circulating leukocytes respond to inflammatory signals by rapid changes of cell adhesive properties.These include cell tethering, rolling, arrest, and firm adhesion, all of which are well described steps of leukocyte recruitment to the sites of inflammation (1). Leukocyte arrest and firm adhesion are mediated exclusively by integrin receptors (2). At the same time integrins can also mediate tethering and rolling (3). These largely diverse cell adhesive properties are achieved by sophisticated conformational regulation; multiple states of the same molecule with different affinity for its ligand and different degrees of molecular unbending are attributed to various types of "cellular behavior." It is proposed that the low affinity bent state translates into a non-adhesive resting cell, the low affinity unbent or extended state of integrin results in cell rolling, and the high affinity state promotes cell arrest (4, 5). However, the exact sequence of conformational events and the relationship between integrin conformational and functional activity remain key questions (6).Integrin conformation is regulated through G-protein-coupled receptors by a signaling pathway which is initiated by ligand binding to a GPCR, 3 propagated inside the cell, and results in the binding of signaling proteins (such as talin and others) to cytoplasmic domains ...

Conformational Equilibrium of Human Platelet Integrin Investigated by Three-Dimensional Electron Cryo-Microscopy

“…Studies in which FRET was used to study conformational shifts upon activation of integrin α 4 β 1 (Chigaev et al 2003) and α IIb β 3 (Coutinho et al 2007) on the cell surface argue against a mechanistic connection between extension and activation. This conclusion is also supported by studies in which the conformation of α 4 β 1 was probed with antibodies (Chigaev et al 2009). Finally, rotary shadowed images of constitutively inactive integrin α 5 β 1 reveal extended conformers (Takagi et al 2003), a finding that also dissociates the connection between activation and extension.…”

Conformational mAb as a Tool for Integrin Ligand Discovery

“…Thus, ligand binding by itself may mechanically induce a series of conformational changes, since ligand-induced binding of HUTS-21 can be observed on ice. 26 Thus, the exposure of the HUTS epitope appears to refl ect a ligand-induced binding site (LIBS).…”

CD29 expressed on plasma cells is activated by divalent cations and soluble CD106 contained in the bone marrow plasma: refractory activation is associated with enhanced proliferation and exit of clonal plasma cells to circulation in multiple myeloma patients

“…This confirms more recent studies reporting that SDF-1a increases binding to FN without the appearance of activation epitopes on CD29 molecule. 26 These observations show that there are at least two signaling pathways that might regulate the activation of CD49d/CD29 on BM PC. Although it is possible that both classes of signals are required to generate sufficient numbers of activated integrins, their relative contribution to the total active structure is uncertain.…”