Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4

J Biol Chem. 2011 Jun 10;286(23):20375-86. doi: 10.1074/jbc.M110.206185. Epub 2011 Apr 22.


Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gα(i)-coupled G protein-coupled receptors (GPCRs) and deactivated by Gα(s)-coupled GPCRs. The effects of Gα(s)-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescent Dyes / chemistry
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Humans
  • Integrin alpha4beta1 / agonists
  • Integrin alpha4beta1 / metabolism*
  • Leukocyte Rolling / physiology*
  • Lymphocyte Function-Associated Antigen-1 / metabolism*
  • Protein Binding
  • Receptors, G-Protein-Coupled / metabolism*
  • U937 Cells


  • Fluorescent Dyes
  • Integrin alpha4beta1
  • Lymphocyte Function-Associated Antigen-1
  • Receptors, G-Protein-Coupled
  • GTP-Binding Protein alpha Subunits, Gi-Go