Research Highlights

Cell proliferation: Rafting through the lattice

Mgat5^+/+ and Mgat5^-/- cells either untreated (top and bottom) or pretreated (middle) with lactose were immunofluorescently labeled for EGFR (red) and Cav1 (green). Yellow spots indicate where EGFR and Cav1 colocalize. From Lajoie et al. 2007. © 2007 The Rockefeller University Press.

The cell membrane is subdivided into structurally different domains that determine cell growth and signaling. Among these domains are lipid raft microdomains that are rich in glycolipids, including the lactosamine-containing GM1 glycolipid. A sub-set of these lipid rafts contain caveolin oligomers that endocytose growth receptors such as the epidermal growth factor receptor (EGFR), thereby abating cell growth and proliferation. Not surprisingly, the tumor suppressor locus to which the caveolin-1 (Cav1) gene maps is frequently deleted in human carcinomas.

In addition to membrane lipid bilayer structures, the actin cytoskeleton and the extracellular membrane-bound galectin-glycan lattice (GGL) also influence cell receptor signaling. Formation of cell surface lattices consisting of galectins and their -galactoside ligands is facilitated by the multimeric structure of galectins — such as the pentamers formed by galectin-3. Growth- and leukocyte-signaling receptors are intertwined with the GGL by their N-glycans. This lattice (see also here and here) restricts the lateral mobility of receptors in resting cells that have minimal membrane turnover, and retains receptors at the cell surface in dividing cells with a rapid membrane turnover. As well as galectin abundance, the strength of the GGL is proportional to the number of -galactoside ligands at the ends of branched N-glycans. Ligand number, in turn, increases with the number of attached N-glycans and their modification by the sequential action of four N-acetylglucosaminyltransferases (Mgat1, 2, 4 and 5). Four new studies have now expanded our knowledge about the GGL and cell membrane microdomain organization.

In resting T cells, Lck (leukocyte-specific protein tyrosine kinase) phosphorylation at the activating residue Y394 is blocked by the T-cell transmembrane phosphatase CD45. Chen et al., reporting to the Journal of Biological Chemistry, noticed that in resting Mgat5^-/- T cells Lck Y394 phosphorylation is markedly increased relative to wild-type mice. Y394 phosphorylation also increased when resting T cells were treated with an inhibitor of N-glycan synthesis or with lactose, which disrupts galectin binding to N-glycans. These findings suggest that the GGL regulates Lck activation in the absence of a presented antigen.

When Chen et al. analyzed the membrane organization of resting T cells they found that the GGL counteracted the actin cytoskeleton and promoted partition of CD45 inside, and of the T-cell receptor (TCR)/CD4-Lck complex outside, GM1-enriched microdomains (GEM). When the GGL was disrupted, the TCR/CD4-Lck complex associated with effector molecules connected to the actin cytoskeleton, leading to transfer of the complex to GEMs and Lck activation and, concurrently, actin removal of CD45 from GEMs. These results indicate that a disruption of the GGL is critical in early TCR signaling. Taken together, this study provides a molecular mechanism for coupling microdomain organization on the outer and inner cell membrane leaflets via interaction of galectins to extracellular N-glycans and cytoplasmic domain tethering to actin microfilaments, respectively.

Chen et al. hypothesize that in wild-type T cells a ligand's affinity to the TCR must exceed the strength of the GGL bonds in order to initiate TCR signaling. Interestingly, Mgat5^-/- mice – which possess a weakened GGL - are prone to autoimmune conditions, indicating enhanced TCR signaling. In the Journal of Biological Chemistry, Lee et al. show that the susceptiblity of inbred mouse strains to experimental autoimmune encephalomyelitis (EAE) - an animal model of multiple sclerosis (MS) - correlates with an inherited deficiency in the level of Mgat expressed. In the EAE-susceptible PL/J mouse strain, Mgat deficiency promoted a spontaneous MS-like disease. The authors hypothesize that in addition to the influence of a specific major histocompatibility complex haplotype, EAE may also be caused by weakened GGL leading to enhanced TCR signaling.

Lajoie et al. have shown in the Journal of Cell Biology that receptor recruitment to the GGL competes with negatively regulating Cav1 domains, thereby promoting tumor cell EGFR signaling. Tumor cells extracted from Mgat5^-/- mice expressing a viral oncogene were markedly less sensitive to EGF, lacked the epithelial-mesenchymal transition and had markedly reduced levels of caveolin. Mgat5 re-expression in Mgat5^-/- cells restored Cav1 levels to normal, which indicates that Mgat5 expression and glycan synthesis influence Cav1 expression. A subgroup of Mgat5^-/- cells exhibited a greater reduction in caveolin levels than typical Mgat5^-/- cells, and Mgat5 re-expression failed to restore Cav1 to normal levels. This indicates that further genetic factors may prevent the full expression of Cav1 even in the presence of Mgat5.

Lajoie et al. found that even though the GGL and Cav1 domains independently restrict lateral movement of EGFR in the plasma membrane, in Mgat5^-/- mice Cav1 levels do not affect the lateral diffusion of EGFR. These findings indicate that Mgat5 overexpression — a common feature of cancer cells — can override the tumor suppressor function of Cav1 by recruiting EGFR to the GGL and away from the negatively regulating Cav1 domains.

Interestingly, in an independent study by Tanikawa et al. the interaction between galectin-9 and GM1 was found to induce clustering of lipid rafts. The association between c-Src/ERK (extracellular signal-regulated kinase) and lipid rafts causes local c-Src/ERK and ligand concentrations to increase — a prerequisite for osteoblast differentiation. Thus, this study contributes to the evidence for GGL significance in cell development provided by the other three studies presented here.

Author: Mirko von Elstermann