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

S. Oliferenko, K. Paiha, T. Harder, V. Gerke, C. Schwärzler, H. Schwarz, H. Beug, U. Günthert, L. A. Huber
CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergentresistant 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
more » ... questration 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 ob-served. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTP ␥ S or phalloidin in a semipermeabilized and cholesteroldepleted 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 detergentinsoluble 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. Key words: CD44 • annexin II • epithelial cell line • lipid rafts • cytoskeleton L IPIDS and lipid derivatives participate in signal transduction events occurring on the plasma membrane, and regulate plasma membrane-cytoskeleton interactions (Chong et al., 1994; Sun et al., 1995; Hirao et al., 1996) . Moreover, recent developments put forward a concept that certain lipids can organize plasma membrane microdomains, called lipid rafts, representing high-order structures. These microdomains are thought to exist as liquid-ordered phases, characterized by a high degree of acyl chain order, which contain sphingolipids with their predominantly saturated hydrocarbon tails and cholesterol (see reviews Brown and London, 1998; Ikonen and Simons, 1998; Brown, 1998) . The intervening regions are en-visioned to be in liquid-disordered membrane phase occupied by unsaturated phosphatidyl-choline molecules. Sphingolipid-cholesterol rafts are relatively insoluble in nonionic detergents (e.g., Triton X-100) on ice, and can be recovered together with certain membrane proteins as insoluble complexes called detergent-insoluble glycolipidenriched domains (DIGs) 1 (Parton and Simons, 1995) . DIGs can be biochemically distinguished from detergentinsoluble cytoskeleton-associated material by density gradient centrifugation, where, due to their high lipid content,
doi:10.5451/unibas-ep6527 fatcat:odmxgylfdrbzbkotlw4lg33vn4