Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin.

Abstract

Abstract. It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4°C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipidprotein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPIanchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250. We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPIanchored proteins, ot and fl subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes. diverse group of proteins is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) a anchor (reviewed by Cross, 1990;Englund, 1993). This group includes lymphocyte and trypanosome surface antigens, adhesion molecules, exofacial enzymes, and receptors. The core structure of the anchor consists of a phosphoethanolamine residue amide-linked to the COOH-terminal amino acid of the protein, three mannose residues, an unacetylated glucosamine residue, and a phosphatidylinositol molecule that is embedded in the outer leaflet of the lipid bilayer. This core is transferred en bloc to the polypeptide chain in the endoplasmic reticulum, following cleavage of a hydrophobic COOH-terminal domain that serves as a signal for anchor addition.

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

“…Caveolaedependent endocytosis also requires intact lipid rafts and hence, is normally inhibited by cholesterol depletion (Murata et al, 1995). Caveolin is a key component of caveolae (Morrow & Parton, 2005); however, a number of previous studies have shown that RAW264.7 cells lack caveolin (Cameron et al, 1997;Fra et al, 1994;Gorodinsky & Harris, 1995;Lyden et al, 2002) and we have confirmed these observations in our RAW264.7 cells. These data do not rule out the possibility that MNV-1 infection may occur through a caveolae-dependent pathway in other cell types that express caveolin.…”

“…Therefore, we investigated the role of caveolae in MNV-1 infection. Caveolae formation requires caveolin (Morrow & Parton, 2005); however, a number of reports have shown that RAW264.7 cells lack caveolin and therefore caveolae (Cameron et al, 1997;Fra et al, 1994;Gorodinsky & Harris, 1995;Lyden et al, 2002). In order to confirm the absence of caveolin-1 in our RAW264.7 cells, we subjected RAW264.7 cell lysates to immunoblotting.…”

Compartmentalized Activation of the High Affinity Immunoglobulin E Receptor within Membrane Domains

“…15 and as described below), and both contain similar amounts of cellular protein (Ͻ2% of the total). 2 By these criteria, the domains obtained using 0.05% Triton X-100 for cell lysis appear to be identical to other membrane domains previously described that do not contain caveolin (10,(13)(14)(15). Furthermore, the aggregation-dependent association of Fc⑀RI with membrane domains shows selectivity among transmembrane cell surface receptors, as Fc⑀RI but not Type I interleukin-1 receptors, both expressed on Chinese hamster ovary cells, associate with membrane domains following aggregation.…”

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein

“…In this instance APP was shown to interact directly with caveolin, and depletion of the latter protein using antisense oligonucleotides prevented α-secretase cleavage, suggesting that caveolin may have a role in the α-secretase-mediated processing of APP in non-neuronal cells. However, we have detected only a very small proportion of APP in DIGs isolated from COS-7 cells using the same methodology (E. T. Parkin, A. J. Turner and N. M. Hooper, unpublished work), and, as caveolin is absent from neuronal cells [16,27,28], the relevance of this observation to the processing of APP in the brain remains unclear.…”

“…Whilst caveolin is highly expressed in lung and muscle, it is undetectable in other tissues such as spleen, kidney, liver, brain and testis [16]. Despite the absence of caveolin, DIGs can be isolated from neuronal sources and are found to contain a number of GPI-anchored proteins, such as alkaline phosphatase, 5h-nucleotidase, F3 protein, the prion protein [24][25][26][27], tyrosine kinases, and α-and β-subunits of heterotrimeric G-proteins [28,29]. Another protein, flotillin, has been found to be present exclusively in DIGs isolated from neuronal sources and can, therefore, be used in place of caveolin as a transmembrane polypeptide-anchored marker for these membrane domains [30][31][32].…”