Subburaj Ilangumaran

Microdomains in lymphocyte signalling: beyond GPI-anchored proteins

Abstract Plasma membrane microdomains that are enriched in sphingolipids, cholesterol and glycosylphosphatidylinositol-anchored proteins appear to be important in T-cell signalling. Here, Subburaj Ilangumaran, Hai-Tao He and Daniel Hoessli discuss the possible mechanisms involved in T-cell activation via such microdomains.

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners [1] and temporary elaboration of supramolecular structures [2], to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptors cytoplasmic domains and intracellular signaling and adaptor proteins [3]. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptors cytoplasmic domains, an event that initiates the signaling cascade [4]. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular non-receptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor [5]. The concept of the signaling platform [6] has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling.The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane [7,8]. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact [9]. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.

Signal Transduction via CD44: Role of Plasma Membrane Microdomains

CD44 is the principal cell surface receptor for extracellular matrix glycosaminoglycan hyaluronan. CD44-hyaluronan mediated cell adhesion is important in several pathophysiological processes such as inflammation and metastatic spread of cancer cells. It has been recently recognized that CD44 also functions as a signaling receptor in a variety of cell types. Cell stimulation by monoclonal anti-CD44 antibody or natural CD44 ligands activate several signaling pathways that culminate in cell proliferation, cytokine secretion, chemokine gene expression and cytolytic effector functions. One of the earliest signaling events following stimulation via CD44 is tyrosine phosphorylation of intracellular proteins substrates, and CD44 mediated cellular activation could be abolished by protein tyrosine kinase (PTK) inhibitors. The Src-family non-receptor PTKs such as Lck, Fyn, Lyn and Hck were shown to be coupled to CD44 via sphingolipid-rich microdomains (lipid rafts) of the plasma membrane. Studies on T cell receptor and IgE receptor mediated signaling in lymphocytes and mast cells have consolidated the notion that microdomains consist of signaling platforms where components of multiple signaling pathways are assembled. Co-isolation of CD44 with microdomains strongly suggests that CD44 generates cellular activation signals utilizing the signaling machinery of the plasma membrane microdomains.

Transfer of exogenous glycosylphos-phatidylinositol (GPI)-linked molecules to plasma membranes

Purified GPI-linked molecules incorporate spontaneously in vitro into mammalian cell plasma membranes. Recent evidence suggests that the transferred molecules insert stably into the external leaflet of the acceptor cell plasma membrane through their acyl chains and behave subsequently in a way similar to endogenous GPI-linked molecules. Transfer of GPI-linked proteins between cells has also been documented in vivo and may explain the uptake by host cells o f pathogen-derived virulence factors carrying a GPI anchor. In this comment article, Subburaj Ilangumaran, Peter Robinson and Daniel Hoessli review what is known about GPI transfer and discuss the use of GPI transfer for transient cell-surface expression of foreign proteins.

Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling.

Abstract.Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose- capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-γ) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production.

Distinct interactions among GPI-anchored, transmembrane and membrane associated intracellular proteins, and sphingolipids in lymphocyte and endothelial cell plasma membranes

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins are enriched in sphingolipid-rich plasma membrane domains, which are often isolated as low-density membrane complexes. This association is believed to arise from the interactions between the GPI-acyl chains and sphingolipids, but is not fully understood. In this study, we compared the physical properties of GPI-anchored glycoproteins from a non-polarized (murine T-lymphocyte) and a polarized (human endothelial) cell by equilibrium density gradient centrifugation after extraction by detergents under identical conditions. Unlike those on epithelial cells, the GPI-anchored proteins of lymphocytes (Thy-1 and the heat stable antigen CD24) were enriched in the floating fractions after extraction over a wide range of octylglucoside concentrations. In contrast, the floatability of endothelial GPI-anchored CD59 was markedly diminished, not only by octylglucoside, but also by increasing concentrations of Triton X-100. Distribution of cholera toxin binding ganglioside GM1 in the sucrose gradient fractions closely followed that of the GPI-anchored proteins in both lymphocytes and endothelial cells under most extraction conditions. Analysis of the intracellular acylated molecules revealed that a significant amount of p56(lck) was always associated with the floating GPI-rich fractions of lymphocytes when extracted by Triton X-100 or octylglucoside at 4 degrees C, while the behaviour of endothelial cell caveolin was comparable to that of CD59. The transmembrane glycoproteins CD45 in lymphocytes and MHC class I antigen in endothelial cells interacted weakly with GPI domains, whereas endothelial CD44 and lymphocyte CD26 displayed a strong association. These results show that: (1) the physical properties of different GPI-anchored proteins may vary significantly; and (2) transmembrane and acylated intracellular proteins could be associated with GPI domains to a variable extent. These differences probably reflect cell type-specific interactions of GPI anchors with the sphingolipid framework of plasma membranes, as well as extracellular interactions of GPI-anchored glycoproteins with neighbouring cell surface molecules.

A Positive Regulatory Role for Suppressor of Cytokine Signaling 1 in IFN-γ-Induced MHC Class II Expression in Fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1−/− mice due to unbridled IFN-γ signaling. We used mouse embryo fibroblasts derived from SOCS1−/− mice to investigate the role of SOCS1 in IFN-γ signaling pathways. SOCS1−/− fibroblasts were exquisitely sensitive to the IFN-γ-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1−/− fibroblasts were inefficient in MHC class II surface expression following IFN-γ stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-γ in SOCS1−/− fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-γ-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-γ-induced MHC class II expression in SOCS1−/− cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-γ-induced MHC class II proteins in fibroblasts.