Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Abstract

We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquidordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoylor dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaffolding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that crosslinking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.

Clustering of Raft-Associated Proteins in the External Membrane Leaflet Modulates Internal Leaflet H-Ras Diffusion and Signaling

“…However, the existence of such domains in living cells has recently been debated (1,8,10,17,26,33,44,49,53,54). Cholesterol-sensitive assemblies were suggested to exist in both the external and internal leaflets of the plasma membrane (7,18,26,44,50,53,55), but the mechanisms that couple them to enable transbilayer effects on signaling remained elusive.…”

“…Related studies were conducted mainly in T cells, where external clustering of raftassociated molecules was reported to induce intracellular signaling, interpreted to indicate a role for raft stabilization/ growth in signal transduction (9,20,21,37,45,53). However, even in these cells, which are characterized by high spatial organization at the immunological synapse, the involvement of lipid rafts in clustering-induced transbilayer signaling was recently questioned (8,15,29,54).Here, we employed fluorescence recovery after photobleaching (FRAP) to study the effects of clustering outerleaflet and transmembrane influenza hemagglutinin (HA) mutants that differ in raft association on the interactions of Ras isoforms with inner-leaflet raft domains and on Ras signaling. Ras proteins are small GTPases which operate as molecular switches at the inner leaflet and regulate proliferation, differentiation, and cell survival (18, 46).…”

“…In spite of a wealth of studies on the functional importance of interactions with raft-like domains for many cellular processes (reviewed in references 17, 18, 26, 49, 50, and 53), many questions remain concerning the size, lifetime, and even the existence of lipid rafts in live cells (1,8,10,17,18,23,26,27,33,45,49,51,54). The original simplistic view of lipid rafts was as preexisting liquid-ordered domains enriched in cholesterol and sphingolipids, into which specific proteins partition preferentially (4, 10, 49).…”

Anti–Prostate Stem Cell Antigen Monoclonal Antibody 1G8 Induces Cell Death In vitro and Inhibits Tumor Growth In vivo via a Fc-Independent Mechanism

“…3 One hint to PSCA's function may come from its structure as a GPI-anchored protein. GPI-anchored proteins characteristically localize to lipid rafts, detergent-resistant membrane microdomains believed to play a role in integrating signal transduction pathways (14)(15)(16). Several antibodies targeting GPIanchored and raft-associated proteins have been reported to have biological activity, including antibodies against CD24 (GPIanchored), CD20 (raft-associated), and CD48 (GPI-anchored).…”

The Effect of Serum on the PEG-Based Crosslinker-Induced Spheroid Formation of Pancreatic β-Cell

“…Using hydrophobic interaction, we believe that cell membrane of different cells can be anchored without causing any damage to the structure. The hydrophobic unit such as lipid, e.g., cholesterol and oleyl chain can anchor to phospholipids bilayer of lipid membranes such as liposomes and cells [9][10][11][12][13][14][15][16][17][18]. The designed polymeric crosslinker consists of two distinct units: Hydrophobic unit which can anchor to phospholipids bilayer of cell membrane, and a hydrophilic unit which promotes water solubility to utilize in aqueous media.…”