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

Alexandre Chigaev, Yelena Smagley, Yinan Zhang, Anna Waller, Mark K. Haynes, Or Amit, Wei Wang, Richard S. Larson, Larry A. Sklar

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

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 Gαi-coupled G protein-coupled receptors (GPCRs) and deactivated by Gαs- coupled GPCRs. The effects of Gαs-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.

Original languageEnglish (US)
Pages (from-to)20375-20386
Number of pages12
JournalJournal of Biological Chemistry
Volume286
Issue number23
DOIs
StatePublished - Jun 10 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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