Oliferenko S, Paiha K, Harder T, Gerke V, Schwärzler C, Schwarz H, Beug H, Günthert U, Huber L A
IMP, Research Institute of Molecular Pathology, A-1030 Vienna, Austria.
J Cell Biol. 1999 Aug 23;146(4):843-54. doi: 10.1083/jcb.146.4.843.
CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.
CD44是透明质酸（HA）的主要细胞表面受体，已证实在成纤维细胞和血细胞中，它定位于抗去污剂的富含胆固醇的微结构域，即所谓的脂筏。在此，我们研究了极化乳腺上皮细胞基底外侧膜平面内CD44的分子环境。我们发现CD44可分配到其细胞质面含有膜联蛋白II的脂筏中。通过隔离质膜胆固醇，CD44和膜联蛋白II都从这些脂筏中释放出来。通过活细胞中的交联实验证明膜联蛋白II和CD44分配到同一类型的脂筏中。首先，当抗CD44抗体使CD44在细胞表面聚集时，膜联蛋白II被招募到CD44聚集区的细胞质小叶中。其次，膜联蛋白II的反式显性突变体的表达导致细胞内、膜下膜联蛋白II-p11聚集体的形成，从而导致CD44共聚集。此外，还观察到肌动蛋白束频繁重定向到这些聚集区。在半透性和胆固醇耗尽的细胞系统中，添加GTPγS或鬼笔环肽可稳定这些基底外侧CD44/膜联蛋白II-脂筏复合物。用光漂白后荧光恢复（FRAP）评估，CD44在质膜中的低侧向流动性取决于质膜胆固醇的存在和完整的肌动蛋白细胞骨架。肌动蛋白细胞骨架的破坏显著增加了可从轻去污剂不溶性膜组分中回收的CD44的比例。综上所述，我们的数据表明，在乳腺上皮细胞中，绝大多数CD44以胆固醇依赖的方式与脂筏中的膜联蛋白II相互作用。这些含有CD44的脂质微结构域与下面的肌动蛋白细胞骨架相互作用。
