The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes

Y van Kooyk, S J van Vliet, C G Figdor

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)26869-77
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number38
Publication statusPublished - 17 Sep 1999

Cite this

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title = "The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes",
abstract = "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.",
keywords = "Actins/metabolism, Animals, Cell Adhesion, Cells, Cultured, Cytoskeleton/metabolism, Intercellular Adhesion Molecule-1/metabolism, Ligands, Lymphocyte Function-Associated Antigen-1/genetics, Mutagenesis, Site-Directed, Protein Binding, Protein Conformation, Structure-Activity Relationship",
author = "{van Kooyk}, Y and {van Vliet}, {S J} and Figdor, {C G}",
year = "1999",
month = "9",
day = "17",
language = "English",
volume = "274",
pages = "26869--77",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes. / van Kooyk, Y; van Vliet, S J; Figdor, C G.

In: Journal of Biological Chemistry, Vol. 274, No. 38, 17.09.1999, p. 26869-77.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes

AU - van Kooyk, Y

AU - van Vliet, S J

AU - Figdor, C G

PY - 1999/9/17

Y1 - 1999/9/17

N2 - 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.

AB - 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.

KW - Actins/metabolism

KW - Animals

KW - Cell Adhesion

KW - Cells, Cultured

KW - Cytoskeleton/metabolism

KW - Intercellular Adhesion Molecule-1/metabolism

KW - Ligands

KW - Lymphocyte Function-Associated Antigen-1/genetics

KW - Mutagenesis, Site-Directed

KW - Protein Binding

KW - Protein Conformation

KW - Structure-Activity Relationship

M3 - Article

VL - 274

SP - 26869

EP - 26877

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 38

ER -