title: Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4.
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 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.
authors.affiliation: Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. achigaev@salud.unm.edu
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chigaev
authors.forename: Yelena
authors.initials: Y
authors.lastname: Smagley
authors.forename: Yinan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: Mark K
authors.initials: MK
authors.lastname: Haynes
authors.forename: Or
authors.initials: O
authors.lastname: Amit
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Richard S
authors.initials: RS
authors.lastname: Larson
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000819:agonists / Q000378:metabolism*; D036904:Leukocyte Rolling / Q000502:physiology*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D039041:Integrin alpha4beta1; D016169:Lymphocyte Function-Associated Antigen-1; D043562:Receptors, G-Protein-Coupled; D019206:GTP-Binding Protein alpha Subunits, Gi-Go