Elsevier

Cellular Signalling

Volume 24, Issue 8, August 2012, Pages 1641-1647
Cellular Signalling

Raft coalescence and FcγRIIA activation upon sphingomyelin clustering induced by lysenin

https://doi.org/10.1016/j.cellsig.2012.04.007Get rights and content

Abstract

Activation of immunoreceptor FcγRIIA by cross-linking with antibodies is accompanied by coalescence of sphingolipid/cholesterol-rich membrane rafts leading to the formation of signaling platforms of the receptor. In this report we examined whether clustering of the raft lipid sphingomyelin can reciprocally induce partition of FcγRIIA to rafts. To induce sphingomyelin clustering, cells were exposed to non-lytic concentrations of GST-lysenin which specifically recognizes sphingomyelin. The lysenin/sphingomyelin complexes formed microscale assemblies composed of GST-lysenin oligomers engaging sphingomyelin of rafts. Upon sphingomyelin clustering, non-cross-linked FcγRIIA associated with raft-derived detergent-resistant membrane fractions as revealed by density gradient centrifugation. Pretreatment of cells with GST-lysenin also increased the size of detergent-insoluble molecular complexes of activated FcγRIIA. Sphingomyelin clustering triggered tyrosine phosphorylation of the receptor and its accompanying proteins, Cbl and NTAL, in the absence of receptor ligands and enhanced phosphorylation of these proteins in the ligand presence. These data indicate that clustering of plasma membrane sphingomyelin induces coalescence of rafts and triggers signaling events analogous to those caused by FcγRIIA activation.

Highlights

► Binding of lysenin to the plasma membrane induces clustering of raft sphingomyelin. ► Upon sphingomyelin aggregation, non-activated FcγRIIA associates with rafts. ► Sphingomyelin clustering triggers phosphorylation of FcγRIIA and signaling proteins. ► Complexes of activated FcγRIIA coalesce after sphingomyelin clustering. ► Raft merging evoked by sphingomyelin aggregation induces signaling of FcγRIIA.

Introduction

Numerous proteins and lipids in the plasma membrane display heterogeneous lateral distribution important for signal transduction and membrane trafficking. Among them, assemblies of sphingolipids and cholesterol, named rafts, have drawn special attention. The existence of rafts was first verified in model membranes. It was shown that sphingolipids acylated with long saturated fatty acids interact with cholesterol and form liquid ordered phase, separating from liquid disordered unsaturated glycerophospholipids. The dense packing of lipids renders sphingolipid- and cholesterol-enriched microdomains insoluble in cold non-ionic detergents, like Triton X-100 [1], [2]. However, in the plasma membrane, the biochemical milieu for raft formation is more complex due to the abundance of various lipid/lipid, lipid/protein and protein/protein lateral interactions [3], [4]. Nevertheless, ample recent data indicate that in unstimulated cells rafts exist as nano-scale complexes of sphingolipids, cholesterol and distinct proteins, including glycosylphosphatidylinositol (GPI)-anchored proteins, palmitoylated cytosolic proteins and certain transmembrane proteins. Such assemblies have a subsecond life-time but can coalesce into larger more stable platforms upon activation of distinct plasma membrane receptors [5], [6]. Activated Fcγ receptor IIA (FcγRIIA) and other immunoreceptors have been shown to associate with rafts and induce formation of raft-based platforms enabling phosphorylation of the receptors by tyrosine kinases of the Src family anchored in the rafts [7], [8], [9]. Phosphorylation of conserved tyrosine residues of FcγRIIA triggers signaling pathways, including tyrosine phosphorylation of downstream proteins, like Cbl and NTAL adaptors, that eventually leads to immune responses [10]. Under experimental conditions, FcγRIIA activation and clustering can be also induced by cross-linking the receptor with antibodies. Association of cross-linked or ligand-activated FcγRIIA with rafts is reflected by gained resistant of the protein to Triton X-100 extraction and flotation in density gradients [8], [11]. This redistribution of activated FcγRIIA has also been demonstrated in large sheets of native plasma membrane examined under confocal and electron microscopy [12], [13].

The current model of FcγRIIA activation attributes the active role in the raft coalescence to the receptor but predicts a concomitant reorganization of raft lipids. It remains an open question whether clustering of raft lipids could reciprocally affect lateral distribution of FcγRIIA and induce its partition to raft-based platforms. The engagement of sphingomyelin in raft organization makes it a good candidate for such studies. Sphingomyelin is composed of a long-chain sphingoid base, an amide-linked chain of long and saturated fatty acid and a phosphorylcholine head group. It is located mainly in the outer leaflet of the plasma membrane. The content of sphingomyelin in rafts exceeds by 20–30% that in the bulk of the plasma membrane [14], [15]. Clustering of sphingomyelin can be induced by lysenin, a 296 amino acid-long earthworm toxin which selectively targets the lipid recognizing all the structural elements of sphingomyelin [16], [17], [18]. Upon sphingomyelin binding, lysenin forms stable hexamers detectable by SDS-PAGE and electron microscopy [19], [20], [21]. Oligomerization of lysenin is facilitated by cholesterol indicating that sphingomyelin- and cholesterol-rich rafts are sites of preferred binding of lysenin in the plasma membrane [22], [23]. Since a single lysenin molecule binds 5–6 sphingomyelin molecules [24], a lysenin hexamer could induce significant local concentration of the lipid, possibly triggering raft coalescence.

In the present study we examined the effect of binding of recombinant GST-lysenin to the plasma membrane of living cells on the association of FcγRIIA with the raft fraction and receptor phosphorylation. We found that lysenin binding triggers these early events of FcγRIIA activation in a similar manner as does cross-linking of the receptor with antibodies.

Section snippets

Cells

U937 human monocytic cells and transfected baby hamster kidney cells stably expressing FcγRIIA (BHK-FcRII) were obtained and cultured as described previously [9].

Preparation of GST-lysenin

cDNA of lysenin was obtained as described previously [20] and subcloned into glutathione-s-transferase (GST)-encoding pGEX-4T vector [23]. Escherichia coli was transformed with the plasmid and the recombinant protein GST-lysenin was purified on a glutathione-agarose column according to manufacturer's instructions (Sigma).

Lytic activity of GST-lysenin against U937 cells

Cells (1 × 106

GST-lysenin oligomerizes upon binding to sphingomyelin in plasma membrane rafts

Sphingomyelin, glycolipids and cholesterol are enriched in rafts of the plasma membrane which can be isolated as detergent-resistant membrane fragments (DRM) by ultracentrifugation [1]. Due to their enrichment in sphingomyelin these membrane fragments can be detected by GST-lysenin, as revealed by density gradient fractionation of Triton X-100 lysates of U937 cells exposed to the probe (Fig. 1). After fractionation, GST-lysenin was found exclusively in low-density fractions 1 and 2 of the

Discussion

Activation of immunoreceptor FcγRIIA leads to its clustering in the plane of the plasma membrane and formation of distinct plasma membrane structures visible under electron microscope as electron-dense assemblies [12]. The structures are highly enriched in activated, tyrosine-phosphorylated FcγRIIA, GPI-anchored CD55 protein, double-acylated Lyn kinase and palmitoylated transmembrane adaptor protein NTAL. These data, supported by biochemical analysis of Triton X-100-insoluble DRM fractions

Acknowledgments

This work was supported by grant N N401 557040 from the National Science Center, Poland.

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