Role for lipid rafts in regulating interleukin-2 receptor signaling.

Lipid rafts are plasma membrane microdomains characterized by a unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. In this report, the first evidence is provided for a role of these lipid microdomains in regulating interleukin-2 receptor (IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol-anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2-induced proliferation in T cells. IL-2Ralpha is shown to be constitutively enriched in rafts and further enriched in the presence of immobilized anti-Thy-1. In contrast, IL-2Rbeta and IL-2Rgamma, as well as JAK1 and JAK3, are found in soluble membrane fractions, and their localization is not altered by anti-Thy-1. IL-2-mediated heterotrimerization of IL-2R chains is shown to occur within soluble membrane fractions, exclusively, as is the activation of JAK1 and JAK3. As predicted by these results, the disruption of lipid raft integrity did not impair IL-2-induced signaling. Thus, the sequestration of IL-2Ralpha within lipid microdomains restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2Rbeta and IL-2Rgamma.

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2.

Glycosylphosphatidylinositols (GPIs) are attached to the C-termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI-N-acetylglucosaminyltransferase (GPI-GnT), which transfers N-acetylglucosamine from UDP- N-acetylglucosamine to phosphatidylinositol. GPI-GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG-A, PIG-H, PIG-C and GPI1. Here, we report that GPI-GnT requires another component, termed PIG-P, and that DPM2, which regulates dolichol-phosphate-mannose synthase, also regulates GPI-GnT. PIG-P, a 134-amino acid protein having two hydrophobic domains, associates with PIG-A and GPI1. PIG-P is essential for GPI-GnT since a cell lacking PIG-P is GPI-anchor negative. DPM2, but not two other components of dolichol-phosphate-mannose synthase, associates with GPI-GnT through interactions with PIG-A, PIG-C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI-GnT; however, the enzyme activity is enhanced 3-fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI-GnT and imply co-regulation of GPI-GnT and the dolichol-phosphate-mannose synthase that generates a mannosyl donor for GPI.

The function of GPI-anchored proteins in T cell development, activation and regulation of homeostasis.

Many glycosylphosphatidyl-inositol-anchored proteins (GPI-AP) are expressed on T lymphocytes. Ligand or mAb-mediated aggregation of all GPI-AP tested to date results in the initiation of signal transduction pathways via the activation of src family protein tyrosine kinases. Src family kinases co-localise with GPI-AP in specialised sub-domains of the plasma membrane, referred to as detergent insoluble membrane microdomains (DIGS), which are thought to function as signalling platforms. GPI-AP may play a role in the regulation of T cell clonal expansion and effector functions at multiple levels, including the initiation of T cell activation through the antigen receptor complex, the regulation of ongoing responses supported by persisting antigen, as well as proliferative responses to the major T cell growth factor, IL-2. Evidence supporting the role of GPI-AP in the regulation of T cell development, activation and homeostasis is discussed, as well as insights provided by studies in humans and mice lacking GPI-AP.

Immobilization of glycosylphosphatidylinositol-anchored proteins inhibits T cell growth but not function.

Accumulating evidence suggests that proteins tethered to the plasma membrane through glycosylphosphatidylinositol (GPI) anchors share common biological properties. In the present study we demonstrate that GPI-anchored proteins regulate T cell growth. Specifically, anti-TCR-induced proliferation was profoundly inhibited by co-immobilized mAb specific for Thy-1, CD48 and Ly6A/E. However, neither IL-2 production nor the effector function of cytotoxic T lymphocytes was impaired in these circumstances. Analysis of the IL-2 receptor (IL-2R) signaling pathway revealed that the association of IL-2R beta and gamma chains with the Janus kinases, JAK1 and JAK3, was not perturbed in the presence of mAb specific for GPI-linked proteins. However, in these conditions, IL-2-mediated recruitment of IL-2Ralpha, beta and gamma chains, resulting in the formation of the high-affinity hetero-trimeric IL-2R, was inhibited. The resulting phosphorylation of JAK1 and JAK3, indicative of their activation states, was correspondingly reduced. These results characterize a novel state of T cell physiology in which effector function is maintained, in the absence of clonal expansion. A physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function is discussed.

Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T.

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.