Pavalko F M, LaRoche S M
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
J Immunol. 1993 Oct 1;151(7):3795-807.
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 beta-subunit (beta 2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein alpha-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 alpha-actinin is homologous with a region in the cytoplasmic domain of the integrin beta 1-subunit, which also binds alpha-actinin. We demonstrate that the association of alpha-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with alpha-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 alpha-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-alpha. Very little CD18 coprecipitates with alpha-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with alpha-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between alpha-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 alpha-actinin-mediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, alpha-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 alpha-actinin.
Mac-1和LFA-1是白细胞或CD18整合素粘附分子亚家族的成员，在各种激动剂激活中性粒细胞后，它们会迅速从低亲和力状态转变为高亲和力状态。目前对CD18整合素依赖性中性粒细胞粘附的控制以及调节整合素亲和力的机制了解甚少。细胞质结构域缺失实验表明，整合素的细胞质结构域对于整合素的正常功能是必需的，这表明与细胞内蛋白质的相互作用参与其中。我们专注于鉴定与整合素白细胞亚家族共有的β亚基（β2或CD18）的细胞质结构域相互作用的细胞骨架蛋白，该亚家族包括LFA-1、Mac-1和p150,95。在固相结合试验和亲和层析实验中，肌动蛋白结合蛋白α-辅肌动蛋白在体外与对应于CD18细胞质结构域一部分的肽结合。CD18细胞质结构域中与α-辅肌动蛋白结合的肽序列与整合素β1亚基细胞质结构域中的一个区域同源，该区域也与α-辅肌动蛋白结合。我们通过在非变性条件下从受刺激的人中性粒细胞中用α-辅肌动蛋白进行共免疫沉淀CD18，证明α-辅肌动蛋白与CD18的结合在生理上是相关的。使用抗CD18的单克隆抗体探测免疫沉淀复合物的蛋白质印迹，当细胞用趋化肽FMLP或细胞因子白三烯B4或TNF-α激活时，发现CD18与α-辅肌动蛋白共沉淀。未激活的细胞中很少有CD18与α-辅肌动蛋白共沉淀。低至10 nM的FMLP浓度就足以诱导CD18与α-辅肌动蛋白的结合；在用1 nM FMLP激活的细胞中几乎检测不到结合。α-辅肌动蛋白与CD18之间的结合是短暂的，在激活后5-10分钟达到峰值，并在20分钟时降至接近静息水平。在体内，CD18不与踝蛋白或纽蛋白共免疫沉淀。我们得出结论，中性粒细胞的激活导致α-辅肌动蛋白介导的CD18整合素与肌动蛋白丝之间的结合。除了其肌动蛋白束集活性外，α-辅肌动蛋白还具有作为肌动蛋白膜连接分子的主要功能，并且整合素亲和力可能受到与涉及α-辅肌动蛋白的肌动蛋白细胞骨架结合的影响。
