Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.

Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-gamma.

5-aminosalicylic acid (5-ASA) is an antiinflammatory drug widely used in the treatment of inflammatory bowel diseases. It is known to inhibit the production of cytokines and inflammatory mediators, but the mechanism underlying the intestinal effects of 5-ASA remains unknown. Based on the common activities of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands and 5-ASA, we hypothesized that this nuclear receptor mediates 5-ASA therapeutic action. To test this possibility, colitis was induced in heterozygous PPAR-gamma(+/-) mice and their wild-type littermates, which were then treated with 5-ASA. 5-ASA treatment had a beneficial effect on colitis only in wild-type and not in heterozygous mice. In epithelial cells, 5-ASA increased PPAR-gamma expression, promoted its translocation from the cytoplasm to the nucleus, and induced a modification of its conformation permitting the recruitment of coactivators and the activation of a peroxisome-proliferator response element-driven gene. Validation of these results was obtained with organ cultures of human colonic biopsies. These data identify PPAR-gamma as a target of 5-ASA underlying antiinflammatory effects in the colon.

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis.

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12.

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12-CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of "selective blockade" of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatic islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9-39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9-39) was an antagonist of the receptor, inhibiting GLP-1-induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.