ACTIVATION OF HUMAN NEUTROPHILS INDUCES AN INTERACTION BETWEEN THE INTEGRIN-BETA-2-SUBUNIT (CD18) AND THE ACTIN-BINDING PROTEIN ALPHA-ACTININ

Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily o f adhesion molecules, rapidly change from a low avidity to a high avid ity state on activation of neutrophils by various agonists. The contro l of CD18 integrin-dependent neutrophil adhesion and the mechanisms th at regulate integrin avidity are poorLy understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integri ns are necessary for proper integrin function and suggest that interac tions with intracellular proteins are involved. We have focused on ide ntifying cytoskeletal proteins that interact with the cytoplasmic doma in of the beta-subunit (beta2 or CD18) common to the leukocyte subfami ly of integrins, which include LFA-1, Mac-1, and p150,95. The actin bi nding protein alpha-actinin associates in vitro with a peptide corresp onding to a portion of the CD18 cytoplasmic domain in solid phase bind ing assays and affinity chromatography experiments. The peptide sequen ce within the CD18 cytoplasmic domain that binds alpha-actinin is homo logous with a region in the cytoplasmic domain of the integrin beta1-s ubunit, which also binds alpha-actinin. We demonstrate that the associ ation of alpha-actinin with CD18 is physiologically relevant by coimmu noprecipitating CD18 with alpha-actinin from stimulated human neutroph ils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to copre cipitate with alpha-actinin when cells were activated with the chemota ctic peptide FMLP or with the cytokines leukotriene B4 or TNF-alpha. V ery little CD18 coprecipitates with a-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the asso ciation of CD18 with alpha-actinin; very little association was detect ed in cells activated with 1 nM FMLP. The association between alpha-ac tinin and CD18 was transient, peaking 5-10 min after activation and de creasing to near resting levels by 20 min. CD18 did not co-immunopreci pitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an alpha-actinin-mediated association between C D18 integrins and actin filaments. In addition to its actin bundling a ctivity, alpha-actinin has a major function as an actin membrane linke r molecule, and integrin avidity may be affected by an association wit h the actin cytoskeleton involving alpha-actinin.