Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.

SummaryIn allergic diseases, the cytokines interleukin (IL)5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are upregulated and have been proposed to cause blood and tissue eosinophilia by inhibition of eosinophil apoptosis. We demonstrate herein, in freshly isolated human eosinophils, that the IL-3/IL-5/GM-CSF receptor [3 subunit interacts with cytoplasmic tyrosine kinases to induce phosphorylation of several cellular substrates, including the [3 subunit itself. The Lyn and Syk intracellular tyrosine kinases constitutively associate at a low level with the IL-3/IL-5/GM-CSF receptor [3 subunit in human eosinophlls. Stimulation with GM-CSF or IL-5 results in a rapid and transient increase in the amount of Lyn and Syk associated with the IL-3/IL-5/GM-CSF receptor [3 subunit. Lyn is required for optimal tyrosine phosphorylation and activation of Syk. In contrast, Syk is not required for optimal tyrosine phosphorylation and activation of Lyn. These data suggest that Lyn is proximal to Syk in a tyrosine kinase cascade that transduces IL-3, IL-5, or GM-CSF signals. Compatible with this model, both Lyn and Syk are essential for the activation of the antiapoptotic pathway(s) induced through the IL-3/IL-5/GM-CSF receptor [3 subunit in human eosinophils.

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56 lck . Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.

No abstract

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI-and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins. INTRODUCTIONIn contrast to transmembrane glycoproteins, surface molecules inserted into the plasma membrane via a glycosylphosphatidylinositol (GPI) 1 membrane anchor are confined to the outer leaflet of the plasma membrane and do not directly communicate with the cell interior (Low, 1989). However, several GPI-anchored proteins have been shown to be potent signal transducers, because their cross-linking leads to increased protein-tyrosine phosphorylation, calcium fluxes, gene expression, and cell activation and/or proliferation (Robinson, 1991). GPI-anchored proteins such as neurotrophic factor receptors transduce signals by ligand-induced interactions with transmembrane receptor protein-tyrosine kinases (PTKs) (Massague, 1996), whereas other GPI-anchored molecules such as CD87 (uPAR), CD16B (Fc␥RIIIB), and CD14 (lipopolysaccharide receptor) interact with integrins (Petty and Todd, 1996) and appear to signal via integrin-dependent pathways. For most other GPI-anchored proteins, signaling is presumed to require association with sphingolipid microdomains (Romagnoli and Bron, 1997;Stulnig et al., 1997), and their coprecipitation with Src family PTKs has been documented in hematopoietic , epithelial (ShenoyScaria et al., 1992), and neuronal (Zisch et al., 1995;Kunz et al., 1996) cells. The molecular nature of this indirect association between GPI-anchored receptors and Src kinases, however, remains unresolved. Sphingolipid microdomains are thought to consist of clusters of sphingolipids that achieve a liquid-ordered state in the presence of cholesterol (Ahmed et al., 1997;Schroeder et al., 1998) and resist solubilization by nonionic detergents. Such sphingolipid microdomain...