Involvement of Gangliosides in Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule TAG-1 Signaling in Lipid Rafts

Abstract

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.Gangliosides, sialic acid-containing glycosphingolipids (GSLs), 1 are found in the outer leaflet of the plasma membrane of all vertebrate cells and are thought to play functional roles in cellular interactions and the control of cell proliferation (1-4).In the nervous system, where gangliosides are particularly abundant, the species and amounts of gangliosides undergo profound changes during development, suggesting that they may play fundamental roles in this process (5). The accumulation of gangliosides within the neurons in ganglioside storage diseases results in extensive neurite outgrowth (6). Exogenously administered gangliosides accelerate the regeneration of neurons in the central nervous system in vivo after lesioning (7). The addition of exogenous gangliosides to primary cultures of neurons and neuroblastoma cells in vitro stimulates cellular differentiation with concomitant neurite sprouting and extension (8 -10). Glucosylceramide synthesis, the first glycosylation step of GSL synthesis, is required for axonal growth in hippocampal neurons (11) and for embryonic development (12). Transfection of the ganglioside GD3 2 synthase cDNA into neuroblastoma cells induces their cholinergic differentiation and neurite sprouting (13). Finally, mice lacking complex gangliosides exhibit axonal degeneration (14). These data show that gangliosides are involved in neural cell differentiation and brain development. However, the molecular mechanisms underlying the ganglioside-depend...

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

“…It is becoming increasingly clear that membrane microdomains are heterogeneous (29). Recent studies have found that raft isolation based on detergent insolubility produces a more heterogeneous mixture of membrane domains than detergent-free preparations (30). In fact, detection of CD47 in light fractions from gradients of detergent lysates has been shown to be unaltered by cyclodextrin treatment (9), and other studies have reported the existence of similar cyclodextrin-resistant domains (31) (32).…”

Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon–glial interactions

“…In contrast, double-null mice lack both simple and complex gangliosides and, therefore, do not possess any functional binding partners for MAG. Gangliosides also may participate in the organization of membrane domains on axons (27,28) and direct adhesion receptors to discrete areas for optimal interaction with the opposing myelin membrane. Furthermore, elevated levels of lactosylceramides with the concomitant reduction of gangliosides may alter plasma membrane organization and cellular activities, as described in refs.…”

Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm

“…We assume that the 27A antibody effect lies in the cross-linking of raft components as reported for other anti-ganglioside antibodies or other antibodies against raft components. 31,39 The cross-linking could induce oligomerization of nephrin regulated by kinases with affinity for nephrin. The signaling effect of the 27A antibody has been confirmed previously using cultured human peripheral blood mononuclear cells as a model in which the src family kinase, syk, was found to become activated.…”

“…31 Gangliosides are sialic acid-containing glycosphingolipids and, as such, important components of lipid rafts. For the isolation of rafts the buffer conditions were chosen that left the rafts intact.…”