The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.
To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.
van Kooyk
Y
Y
Department of Tumor Immunology, University Hospital Nijmegen St. Radboud, 6525 EX Nijmegen, The Netherlands. Y.vanKooyk@dent.kun.nl
van Vliet
S J
SJ
Figdor
C G
CG
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000199:Actins; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1