Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown t o localize to detergent-resistant cholesterol-rich microdomains, called lip id rafts, in fibroblasts and blood cells, Here, we have investigated the mo lecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipi d rafts that contain annexin II at their cytoplasmic face. Both CD44 and an nexin TT were released from these lipid rafts by sequestration of plasma me mbrane cholesterol. Partition of annexin II and CD44 to the same type of li pid rafts was demonstrated by cross-linking experiments in living cells. Fi rst, when CD44 was clustered at the cell surface by anti-CD44 antibodies, a nnexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Seco nd, 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 c omplexes were stabilized by addition of GTP gamma S or phalloidin in a semi permeabilized and cholesterol-depleted cell system. The low lateral mobilit y of CD44 in the plasma membrane, as assessed with fluorescent recovery aft er photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytos keleton dramatically increased the fraction of CD44 which could be recovere d from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 i nteracts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD C4-containing lipid microdomains interact with the underlying acti n cytoskeleton.