Activation of human neutrophils induces an interaction between the Lntegrin β2-subunit (CD18) and the actin binding protein α-actinin

Fredrick Pavalko, Suzette M. LaRoche

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Abstract

Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the β-subunit (β2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein α-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds α-actinin is homologous with a region in the cytoplasmic domain of the integrin β,-subunit, which also binds α-actinin. We demonstrate that the association of α-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with α-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with α-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-α. Very little CD18 coprecipitates with α-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with α-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between α-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an α-actininmediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, α-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving α-actinin.

Original languageEnglish
Pages (from-to)3795-3807
Number of pages13
JournalJournal of Immunology
Volume151
Issue number7
StatePublished - Oct 1 1993

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Actinin
Microfilament Proteins
Neutrophil Activation
Integrins
Lymphocyte Function-Associated Antigen-1
Actin Cytoskeleton
Peptides
Actins
Neutrophils
Leukocytes
Talin
Vinculin
Leukotriene B4
Cytoskeletal Proteins
Affinity Chromatography
Western Blotting

ASJC Scopus subject areas

  • Immunology

Cite this

Activation of human neutrophils induces an interaction between the Lntegrin β2-subunit (CD18) and the actin binding protein α-actinin. / Pavalko, Fredrick; LaRoche, Suzette M.

In: Journal of Immunology, Vol. 151, No. 7, 01.10.1993, p. 3795-3807.

Research output: Contribution to journalArticle

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abstract = "Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the β-subunit (β2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein α-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds α-actinin is homologous with a region in the cytoplasmic domain of the integrin β,-subunit, which also binds α-actinin. We demonstrate that the association of α-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with α-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with α-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-α. Very little CD18 coprecipitates with α-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with α-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between α-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an α-actininmediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, α-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving α-actinin.",
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T1 - Activation of human neutrophils induces an interaction between the Lntegrin β2-subunit (CD18) and the actin binding protein α-actinin

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N2 - Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the β-subunit (β2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein α-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds α-actinin is homologous with a region in the cytoplasmic domain of the integrin β,-subunit, which also binds α-actinin. We demonstrate that the association of α-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with α-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with α-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-α. Very little CD18 coprecipitates with α-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with α-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between α-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an α-actininmediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, α-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving α-actinin.

AB - Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the β-subunit (β2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein α-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds α-actinin is homologous with a region in the cytoplasmic domain of the integrin β,-subunit, which also binds α-actinin. We demonstrate that the association of α-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with α-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with α-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-α. Very little CD18 coprecipitates with α-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with α-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between α-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an α-actininmediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, α-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving α-actinin.

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