Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. Traditional methods for the biochemical isolation of lipid rafts involve the extraction of cells with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, it is important to develop procedures for the isolation of lipid rafts that do not involve detergent extraction. We report here a simplified method for the purification of detergent-free lipid rafts that requires only one short density gradient centrifugation, but yields a membrane fraction that is highly enriched in cholesterol and protein markers of lipid rafts, with no contamination from nonraft plasma membrane or intracellular membranes. Lipid rafts are low-density plasma membrane domains that are involved in a number of cellular processes, such as trafficking (1) and cell signaling (2-5). A variety of proteins have been shown to be selectively enriched in lipid rafts. These include glycosylphosphatidylinositol (GPI)-anchored proteins (6-10) and dually acylated proteins (11-15) that appear to be targeted to rafts as a result of their posttranslational modification with lipids. Several transmembrane proteins, including flotillin (16), receptor tyrosine kinases (17-19), and G protein-coupled receptors (20-25) have also been shown to be enriched in lipid rafts, although the targeting mechanisms for such proteins are not well defined. Caveolin, the structural protein of the subclass of lipid rafts known as caveolae (26,27), is also typically found in low-density plasma membrane fractions. Together, these proteins are used as markers for lipid rafts during biochemical fractionation procedures designed to isolate these plasma membrane domains.Lipid rafts contain high levels of sphingolipids and cholesterol and probably exist in a liquid-ordered phase (28). Because of the presence of cholesterol and sphingomyelin, as well as the preponderance of saturated acyl chains in lipid rafts (29, 30), the acyl chains in these domains tend to be well ordered and tightly packed. This physical property gives rise to the known ability of lipid rafts to withstand disruption by nonionic detergents (6, 31). The high lipid:protein ratio of such detergent-resistant lipid rafts makes them significantly lower in density than other solubilized membrane proteins and allows them to be isolated from other membrane proteins by centrifugation through density gradients.Early preparations of lipid rafts used 1% Triton X-100 to extract whole cells and the low-density, detergent-resistant material was separated from other solubilized membrane fractions by centrifugation on a 5% to 30% sucrose density gradient (6). Subsequently, lipid rafts have been prepared using a variety of other detergents, including Lubrol WX, Lubrol PX, Brij 58, Brij 96, Brij 98, Nonidet P40, CHAPS,. Although preparations of detergent-resi...

Scatchard analysis of the binding of EGF to its receptor yields concave up plots that indicate the presence of two classes of binding sites. However, how two independent classes of sites arise from the expression of a single EGF receptor protein has never been adequately explained. Using a new analytical approach involving the simultaneous fitting of binding isotherms from cells expressing increasing levels of EGF receptors, we show that 125 I-EGF-binding data can be completely explained by a model involving negative cooperativity in an aggregating system. This approach provides an experimentally determined value for the monomer–dimer equilibrium constant, which, for wild-type EGF receptors, corresponds to ≈50,000 receptors per cell. Therefore, changes in receptor expression within the physiological range can modulate the outcome of a signaling stimulus. Analysis of the L680N-EGF receptor mutant, in which the formation of asymmetric kinase domain dimers is blocked, indicates that the kinase dimers make a substantial energetic contribution to the ligand-independent association of EGF receptor monomers, but are not necessary for negative cooperativity. The model accurately predicts the behavior of receptor mutants, such as the dimerization-defective Y246D-EGF receptor, which exhibit a single class of binding sites and provides a framework for understanding secondary dimer formation and lateral signaling in the EGF receptor family.

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys-->Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.

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