Glycosphingolipid-enriched Membrane Microdomains Research Articles

Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

Lipid Raft-Mediated Regulation of Hyaluronan-CD44 Interactions in Inflammation and Cancer.

Hyaluronan is a major component of the extracellular matrix and plays pivotal roles in inflammation and cancer. Hyaluronan oligomers are frequently found in these pathological conditions, in which they exert their effects via association with the transmembrane receptor CD44. Lipid rafts are cholesterol- and glycosphingolipid-enriched membrane microdomains that may regulate membrane receptors while serving as platforms for transmembrane signaling at the cell surface. This article focuses on the recent discovery that lipid rafts regulate the interaction between CD44 and hyaluronan, which depends largely on hyaluronan’s size. Lipid rafts regulate CD44’s ability to bind hyaluronan in T cells, control the rolling adhesion of lymphocytes on vascular endothelial cells, and regulate hyaluronan- and CD44-mediated cancer cell migration. The implications of these findings for preventing inflammatory disorders and cancer are also discussed.

Flotillins in intercellular adhesion - from cellular physiology to human diseases.

Flotillin 1 and 2 are ubiquitous and highly conserved proteins. They were initially discovered in 1997 as being associated with specific caveolin-independent cholesterol- and glycosphingolipid-enriched membrane microdomains and as being expressed during axon regeneration. Flotillins have a role in a large number of physiopathological processes, mainly through their function in membrane receptor clustering and in the regulation of clathrin-independent endocytosis. In this Commentary, we summarize the research performed so far on the role of flotillins in cell-cell adhesion. Recent studies have demonstrated that flotillins directly regulate the formation of cadherin complexes. Indeed, flotillin microdomains are required for the dynamic association and stabilization of cadherins at cell-cell junctions and also for cadherin signaling. Moreover, because flotillins regulate endocytosis and also the actin cytoskeleton, they could have an indirect role in the assembly and stabilization of cadherin complexes. Because it has also recently been shown that flotillins are overexpressed during neurodegenerative diseases and in human cancers, where their upregulation is associated with metastasis formation and poor prognosis, understanding to what extent flotillin upregulation participates in the development of such pathologies is thus of particular interest, as well as how, at the molecular level, it might affect cell adhesion processes.

Modulation of T cell immune functions by the prostaglandin E2 – cAMP pathway in chronic inflammatory states

Cyclic AMP is the intracellular second messenger for a variety of immunoregulatory inflammatory mediators such as prostaglandin E2, adenosine and histamine that signal to effector T cells from monocytes, macrophages and regulatory T cells. Protein kinase A (PKA) type I localizes to lipid rafts in effector T cells during T cell activation and directly modulates proximal signal events including phosphorylation of C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The PKA-Csk immunoregulatory pathway is scaffolded by the A kinase anchoring protein ezrin, the Csk binding protein phosphoprotein associated with glycosphingolipid-enriched membrane microdomains and the linker protein ezrin/radixin/moesin binding protein of 50 kDa. This pathway is hyperactivated in chronic infections with an inflammatory component such as HIV, other immunodeficiencies and around solid tumours as a consequence of local inflammation leading to inhibition of anti-tumour immunity. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.

Transmembrane Adaptor Proteins in the High-Affinity IgE Receptor Signaling

Aggregation of the high-affinity IgE receptor (FcεRI) initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of spleen tyrosine kinase (Syk). Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs). These proteins are characterized by a short extracellular domain, a single transmembrane domain, and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), linker for activation of X cells (LAX), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG), and growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT)]; engagement of four of them (LAT, NTAL, LAX, and PAG) in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production, and chemotaxis.

Lipid Phase Specific Organisation of the Transmembrane Anchor of Influenza Hemagglutinin

Correct transport and localization of membrane proteins to distinct cellular organelles are crucial for cell growth but also for the infection cycle of enveloped viruses. Viral membrane proteins like hemagglutinin of influenza virus, anchored in the membrane via a single transmembrane domain (TMD), are transported to the plasma membrane and - together with other virus components - recruited to the budding site. Cholesterol and glycosphingolipid-enriched membrane microdomains are considered as assembly and budding sites for enveloped viruses such as influenza virus. Besides a most likely location of a sorting signal within the TMD sequence also the width of the bilayer is important, which is, amongst other factors, controlled by the cholesterol content of the membrane.Using different fluorescent-based approaches we study the impact of membrane hydrophobic thickness and membrane packing properties on the sorting behavior of virus derived TMD peptides containing a Trp residue in its center. The emission λmax depending on the hydrophobicity of the surrounding of the Trp residue and parallax analysis of fluorescence quenching are used to determine the Trp location in the lipid bilayer revealing a transmembrane orientation. Membrane thickness is altered by the use of lipids having different acyl chain length and also by the addition of cholesterol or decane shifting λmax. Incorporation of Rhodamine labeled TMD into giant unilamellar vesicles prepared from lipids with varying length and also from ternary lipid mixtures forming distinct liquid phases allows us to study phase dependent TMD localization by fluorescence microscopy. Typically, in the model system the TMD sorts into the liquid disordered phase in contrary to the raft association of hemagglutinin in cells. To address the latter by a more appropriate system, we used viral lipids for vesicle preparation mimicking the natural environment of the TMD.

The adaptor protein EBP50 is important for localization of the protein kinase A–Ezrin complex in T-cells and the immunomodulating effect of cAMP

We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58+/-7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin-EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin-EBP interaction could be a novel and potent strategy for immunomodulation.

Dual Role of SLP-76 in Mediating T Cell Receptor-induced Activation of Phospholipase C-γ1

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

Harming Bystanders

Irradiation of cells is known to cause damage to neighboring cells that are not directly exposed to the radiation. Such bystander responses are important when considering the potential damage caused by environmental exposure to radiation or to radiation used in cancer treatment. Although it is known that cells whose nuclei are exposed to radiation can trigger the bystander response, Shao et al. show that even radiation delivered to the cytoplasm of a single cell suffices. The bystander response was measured by the increase of micronuclei (a measure of chromosomal damage) in the cell population when single human glioblastoma T98G cells were exposed to single 3He2+ ions. The bystander response was blocked by the addition of a nitric oxide (NO) scavenger to the medium, and disruption of glycosphingolipid-enriched membrane microdomains (GEMs) with filipin also blocked the bystander response, suggesting that NO, produced through a process that requires GEMs, appears to mediate a signal that leads to chromosomal damage in neighboring cells. — NG

Harming Innocent Bystanders

Irradiation of a subpopulation of cells is known to cause damage to neighboring cells that are not directly exposed to the radiation, the so-called bystander response. Bystander responses are important when considering the potential damage caused by environmental exposure to radiation or to radiation used in cancer treatment. Although it is known that cells whose nuclei were exposed to radiation can trigger the bystander response, Shao et al. now show that even radiation delivered to the cytoplasm of a single cell triggers bystander responses. To deliver such a targeted irradiation, the authors used a charged-particle microbeam to expose cultured human glioblastoma T98G cells to a controlled number of 3 He 2+ ions. The bystander response was based on the production of micronuclei (MN) (a measure of chromosomal damage) in the cell population. The abundance of MN increased when a single T98G cell in the population was exposed to a single 3 He 2+ ion, and the bystander response occurred in T98G cultures and in cocultures with human primary fibroblast AG0 cells. The bystander response was blocked by addition of a nitric oxide (NO) scavenger to the medium. Disruption of glycosphingolipid-enriched membrane microdomains (GEMs) with filipin also blocked the bystander response, which suggests that signals from the membrane are important. When a chemical assay for NO production was used, an increase in the number of cells positive for NO increased after a single T98G cell was exposed to a single 3 He 2+ ion through the cytoplasm, and this increase in NO was blocked if cells were first treated with filipin. Thus, NO, produced through a process that requires GEMs in response to cytoplasmic irradiation, appears to mediate a signal that causes chromosomal damage in neighboring cells. C. Shao, M. Folkard, B. D. Michael, K. M. Prise, Targeted cytosplasmic irradiation induces bystander responses. Proc. Natl. Acad. Sci. U.S.A. 101 , 13495-13500 (2004). [Abstract] [Full Text]

Measles virus interacts with and alters signal transduction in T-cell lipid rafts.

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na +/H + exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG–EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice.

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.

Phenotypic Effects of CD3ζ Targeting into Glycosphingolipid-Enriched Membrane Microdomains (GEMs) of T Cells

In the present study we tested whether the forced expression of the CD3ζ chain within detergent-resistant, glycosphingolipid-enriched membrane microdomains (GEMs) will result in a constitutively activated phenotype in human T cells. To this aim, a monomeric recombinant protein (LckSH4-CD3ζ), containing the intracellular part of human CD3ζ chain fused to N-terminal double-acylation motif (SH4 domain) of protein tyrosine kinase Lck, was expressed in Jurkat human T lymphoid cell line and its Lck-negative mutant, J.CaM1.6. The Lck SH4 domain indeed predominantly targeted the chimeric protein into GEMs. In transfectants derived from wild-type Jurkat cells, but not in those derived from the Lck-deficient mutant, the LckSH4-CD3ζ protein was constitutively tyrosine-phosphorylated. Tyrosine phosphorylation of a major Jurkat cell phosphoprotein (pp85) was diminished in the transfectants. However, the transfectants did not exhibit any features of constitutively activated T cells, and their responses to anti-CD3 treatment were very similar to the wild-type Jurkat cells. Thus, the constitutive expression of this form of CD3ζ chain in GEMs is not sufficient for eliciting an activated state in the Jurkat cells.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.