Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation. Academic Article uri icon

start page

  • 14337

end page

  • 14346

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 alpha(4)beta(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.

date/time value

  • 2009

Digital Object Identifier (DOI)

  • 10.1074/jbc.M901178200

PubMed Identifier

  • 19251697

volume

  • 284

number

  • 21

keywords

  • Antibodies
  • Binding Sites
  • Cell Line, Tumor
  • Epitopes
  • Humans
  • Integrin alpha4
  • Integrin alpha4beta1
  • Integrins
  • Kinetics
  • Ligands
  • Models, Molecular
  • Mutant Proteins
  • Protein Conformation
  • Receptors, CXCR4
  • Receptors, Formyl Peptide
  • Time Factors
  • Transfection
  • U937 Cells