Beekman Jeffrey M, van der Poel Cees E, van der Linden Joke A, van den Berg Debbie L C, van den Berghe Peter V E, van de Winkel Jan G J, Leusen Jeanette H W
Immunotherapy Laboratory, Department of Immunology, University Medical Center, Ultrech, The Netherland.
J Immunol. 2008 Mar 15;180(6):3938-45. doi: 10.4049/jimmunol.180.6.3938.
Filamin A, or actin-binding protein 280, is a ubiquitously expressed cytosolic protein that interacts with intracellular domains of multiple receptors to control their subcellular distribution, and signaling capacity. In this study, we document interaction between FcgammaRI, a high-affinity IgG receptor, and filamin A by yeast two-hybrid techniques and coimmunoprecipitation. Both proteins colocalized at the plasma membrane in monocytes, but dissociated upon FcgammaRI triggering. The filamin-deficient cell line M2 and a filamin-reconstituted M2 subclone (A7), were used to further study FcgammaRI-filamin interactions. FcgammaRI transfection in A7 cells with filamin resulted in high plasma membrane expression levels. In filamin-deficient M2 cells and in filamin RNA-interference studies, FcgammaRI surface expression was consistently reduced. FcgammaRI localized to LAMP-1-positive vesicles in the absence of filamin as shown by confocal microscopy indicative for lysosomal localization. Mouse IgG2a capture experiments suggested a transient membrane expression of FcgammaRI before being transported to the lysosomes. These data support a pivotal role for filamin in FcgammaRI surface expression via retention of FcgammaRI from a default lysosomal pathway.
细丝蛋白A，即肌动蛋白结合蛋白280，是一种广泛表达的胞质蛋白，它与多种受体的细胞内结构域相互作用，以控制它们的亚细胞分布和信号传导能力。在本研究中，我们通过酵母双杂交技术和免疫共沉淀证明了高亲和力IgG受体FcγRI与细丝蛋白A之间的相互作用。这两种蛋白在单核细胞的质膜上共定位，但在FcγRI触发后解离。利用细丝蛋白缺陷细胞系M2和细丝蛋白重组的M2亚克隆（A7）进一步研究FcγRI与细丝蛋白的相互作用。在A7细胞中用细丝蛋白转染FcγRI导致质膜表达水平升高。在细丝蛋白缺陷的M2细胞和细丝蛋白RNA干扰研究中，FcγRI的表面表达持续降低。共聚焦显微镜显示，在没有细丝蛋白的情况下，FcγRI定位于LAMP-1阳性囊泡，表明其定位于溶酶体。小鼠IgG2a捕获实验表明，FcγRI在被转运到溶酶体之前有短暂的膜表达。这些数据支持细丝蛋白通过将FcγRI保留在默认的溶酶体途径中，在FcγRI表面表达中起关键作用。
