Co-localization of Rac1 and E-cadherin in human epidermal keratinocytes.

The Rac1 small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell cell junctions in epidermal keratinocytes is thought to be dependent on activity of Rac1. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Rac1 expression vectors and analyzed distribution of Rac1 following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP Rac1 proteins distribute to sites of cell cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Rac1 away from sites of cell cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP Rac1 results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Rac1 with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Rac1 neither disrupts cell cell adhesion nor prevents assembly of new cadherin-based adhesion structures.

Crystal structure and functional dissection of the cytostatic cytokine oncostatin M.

BACKGROUND: The cytokine oncostatin M (OSM) inhibits growth of certain tumour-derived cells, induces proliferation in other cell types (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its mechanism of action is via specific binding to gp130 and either the leukaemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems at the cell surface to form an active signalling complex. RESULTS: We report here the crystal structure of human oncostatin M (hOSM) along with mutagenesis data which map the receptor-binding epitopes of the molecule. The structure was determined to a resolution of 2.2 A and conforms to the haematopoietin cytokine up-up-down-down four-helix bundle topology. The site 2 epitope, responsible for gp130 binding, is centred around Gly120 which forms a 'dimple' on the surface of the molecule located on helices A and C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. CONCLUSIONS: The data presented allow functional dissection of the receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion to structurally equivalent residues at the growth hormone-growth hormone receptor interface, implying that certain key features may underlie recognition across the whole cytokine/receptor superfamily. Conversely, detailed comparison of the available structures suggests that variations on a common theme dictate the specificity of receptor-ligand interactions within the gp130 family of cytokines.

Interleukin-11 signals through the formation of a hexameric receptor complex.

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.

Identification of three distinct receptor binding sites of murine interleukin-11.

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.

The interactions of the cytokine-binding homology region and immunoglobulin-like domains of gp130 with oncostatin M: implications for receptor complex formation.

The receptor gp130 is utilized by cytokines including interleukin 6, leukemia inhibitory factor, oncostatin M, cilary neurotrophic factor and cardiotrophin. It is essential for myocardial development and haematopoiesis during embryogenesis, and its role as a shared signal transducer among different cytokines explains their overlapping biological functions. Although gp130 contains a cytokine-binding homology region (CHR) analogous to the extracellular growth hormone receptor, the complexes that utilize gp130 are not simple dimerizations of receptors around a single cytokine but involve receptor interactions with additional sites on the ligand resulting in higher order complexes. Analysis by surface plasmon resonance of the binding of the immunoglobulin-like and CHR domains of the extracellular portion of gp130 to mutants of the cytokine oncostatin M reveal that the CHR forms the main binding site for oncostatin M by a classical site II interaction, but in addition a second interaction occurs involving the receptor's immunoglobulin-like domain and the cytokine's site III at the N-terminus of the D helix. The implications for complex formation are discussed.

An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M.

The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM). Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signal-transducing subunit shared with interleukin-6 and interleukin-11. The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF. In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands. On cells that express LIF-R and gp130, all LIF-R ligands were antagonized. On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R. Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist. The antagonist is therefore likely to work by preventing receptor oligomerization.

Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor.

Interleukin-11 (IL-11) is a polyfunctional cytokine whose biological actions require a specific IL-11 receptor (IL-11R) and the transmembrane transducer gp130. Here we report the production of a soluble form of the murine IL-11R and demonstrate that it interacts with IL-11 ligand with high affinity. The affinity of IL-11 alone for gp130 is below the level of detection, but a complex of IL-11 and soluble IL-11R interacts with gp130 with high affinity. The addition of soluble IL-11R potentiates the effects of exogenous IL-11 in cells that are normally responsive to IL-11. A biological response to IL-11 can be reconstituted in BAF cells transfected with gp130 by addition of IL-11 and soluble IL-11R. These findings show that the cytoplasmic domain of the IL-11R is not required for the biological effects of IL-11 and that a complex of IL-11 and IL-11R mediates signalling by association with gp130.

Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.

Characterization of the receptor binding sites of human leukemia inhibitory factor and creation of antagonists.

Residues in human leukemia inhibitory factor (hLIF) crucial for binding to both the human LIF receptor (R) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity. The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix, carboxyl terminus of the B-helix, and C-D loop. This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF. The two residues of hLIF that contribute the majority of free energy for hLIF-R binding, Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact. This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor. A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding. Residues in hLIF from both the A- and C-helices are involved in binding the gp130 co-receptor. Abolition of the gp130 binding site in hLIF created antagonists of LIF action.

Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II.

The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.

T-cell receptor beta-chain binding to enterotoxin superantigens.

The last few years have seen an enormous jump in our knowledge and understanding of T-cell activation by superantigens. Clearly, a great number of infectious and parasitic organisms utilize superantigens as part of a strategy to evade the immune response of their host. The ability to modulate superantigen effects will give us new means to fight infections, and the knowledge of T-cell activation that we have gained from study of superantigens will, in turn, allow us to modulate the immune system in new ways.

Two adjacent residues in staphylococcal enterotoxins A and E determine T cell receptor V beta specificity.

The T cell receptor (TCR) V beta-determining region of two bacterial superantigens, staphylococcal enterotoxin A (SEA) and SEE, has been mapped to the COOH-terminal region of SEA and SEE using a panel of recombinant SEA/SEE hybrids. Total TCR V beta mRNA enrichment in human peripheral blood T cell cultures was determined by a novel single-tube amplification technique using a redundant V beta-specific primer. SEA routinely enriched mRNA coding for hV beta 1.1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, and 9.1, while SEE, which is 83% homologous to SEA, enriched hV beta 5.1, 6.3, 6.4, 6.9, and 8.1 mRNA. Exchanging residues 206 and 207 was sufficient to convert in toto the TCR V beta response of human peripheral T lymphocytes. In addition, an SEA-reactive murine T cell line, SO3 (mV beta 17), unresponsive to wild-type SEE responded to SEE-S206N207, while an SEE-specific human T cell line, Jurkat (hV beta 8.1), unresponsive to SEA was stimulated strongly by SEA-P206D207. Exchanging all other regions of SEA and SEE except residues 206 and 207 did little to change the V beta response. Thus, the V beta binding region appears to be a stable, discrete domain localized within the COOH-terminal region that is largely unaffected by the considerable amino acid variability between SEA and SEE. This region may interact directly with TCR V beta.

Enterotoxin residues determining T-cell receptor V beta binding specificity.

Superantigens such as the staphylococcal enterotoxins bind to major histocompatibility complex (MHC) class II molecules and activate T cells through a specific interaction between the V beta region of the T-cell antigen receptor (TCR) and the toxin. The TCR beta-chain alone is sufficient to produce the interaction with the enterotoxin-class II complex. Identification of the regions of enterotoxins that interact with TCR has so far proved equivocal because of difficulties in distinguishing between direct effects on T-cell recognition and indirect effects resulting from alteration of binding to class II. For example, amino-terminal truncations of SEB abrogated T-cell stimulation whereas carboxy-terminal truncation of SEA stopped its mitogenic activity. The most comprehensive study to date, accounting for both enterotoxin binding to class II and enterotoxin interactions with the TCR, identified two functionally important regions for SEB binding to TCR. Although the amino-acid sequences of staphylococcal enterotoxins A and E are 82% identical, they activate T cells bearing different V beta elements. We have assayed the binding of cells coated with these enterotoxins to soluble secreted TCR beta-chain protein and find that V beta 3 binds enterotoxin A but not E, whereas V beta 11 binds enterotoxin but not A. To map the amino-acid residues responsible for these different binding specificities, we prepared a series of hybrids between the two staphylococcal enterotoxins. We report that just two amino-acid residues near the carboxy terminus of the enterotoxins are responsible for the discrimination between these molecules by V beta 3 and V beta 11.(ABSTRACT TRUNCATED AT 250 WORDS)