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.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding.

Alpha-dystroglycan (alpha-DG) is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a posttranslational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Using mass spectrometry- and nuclear magnetic resonance (NMR)-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-DG, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a postphosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the like-acetylglucosaminyltransferase (LARGE) protein. These findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

Binding of Lassa virus perturbs extracellular matrix-induced signal transduction via dystroglycan.

The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan (DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooperates with β1 integrins to control cell-matrix interactions. Here, we investigated whether LASV binding to DG triggers signal transduction, mimicking the natural ligands. Engagement of DG by LASV resulted in the recruitment of the adaptor protein Grb2 and the protein kinase MEK1 by the cytoplasmic domain of DG without activating the MEK/ERK pathway, indicating assembly of an inactive signalling complex. LASV binding to cells however affected the activation of the MEK/ERK pathway via α6β1 integrins. The virus-induced perturbation of α6β1 integrin signalling critically depended on high-affinity LASV binding to DG and DG's cytoplasmic domain, indicating that LASV-receptor binding perturbed signalling cross-talk between DG and β1 integrins.

Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor.

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Telmisartan: a different angiotensin II receptor blocker protecting a different population?

The ONgoing Telmisartan Alone and in combination with Ramipril Global Endpoint Trial (ONTARGET()) showed that the angiotensin II receptor blocker (ARB) telmisartan was as protective as the reference-standard ramipril in a broad cross-section of patients at increased cardiovascular risk, but was better tolerated. Telmisartan has a unique profile among ARBs, with a high affinity for the angiotensin II type 1 receptor, a long duration of receptor binding, a high lipophilicity and a long plasma half life. This leads to sustained and powerful blood pressure lowering when compared with the first marketed ARBs, such as losartan and valsartan. Some pharmacological properties of telmisartan clearly distinguish it from other members of the ARB class and may contribute to the clinical effects seen with telmisartan. A class effect for ARBs cannot be assumed. To date, telmisartan is the only ARB that has been shown to reduce cardiovascular risk in at-risk cardiovascular patients.

Characterisation of heteromers and oligomers in the TNF family of ligands and receptors

TNF family ligands and receptors fulfill a number of functions, mainly in the immune system. For example, the ligands BAFF and APRIL control growth and survival of mature Β cells at various stages of differentiation. TNF family ligands usually form homotrimers, but heteromers have also been described for lymphotoxin α1β2 and for BAFF and APRIL. Interestingly, twenty BAFF homotrimers can assemble into virus-like particles coined BAFF 60-mer, which are superior to BAFF 3-mer regarding their ability to signal in primary Β cells. A screen was performed in 293T cells, by co-transfecting differently tagged ligands, to identify six novel heteromers. The specificity of these novel heteromers, however, did not correspond to that of orphan receptors in the TNFR family. Little is known about heteromers of BAFF and APRIL, in particular their receptor-binding specificity and their ability to signal. A method to produce and purify heteromers of defined stoechiometry was developed, and the resulting reagents were used to demonstrate that BAFF2APRIL, like BAFF, binds to all BAFF receptors - namely BAFFR, TACI and Β CM A -, while APRIL2BAFF and APRIL only binds to TACI and BCMA. Heteromers could signal via their cognate receptors, sometimes as potently and sometimes less potently than homomers, depending on the receptors. A promising system to measure the activity of single-chain homo- and heteromers in vivo was set up: it measures mature Β cell rescue upon administration of single-chain ligands into BAFF-ko mice. To tackle the question of the physiological importance of BAFF 60-mer, a point mutation that prevents assembly of mouse BAFF into 60-mer while retaining its ability to form trimers was identified. This mutation (E247K) was introduced by homologous recombination into mouse embryonic stem cells that are now being used to generate knock-in mice. Results obtained in this work will help to better understand the role of various BAFF and APRIL forms that are elevated in a several autoimmune diseases. - Les ligands et récepteurs de la famille du TNF joue un rôle prédominant dans le système immunitaire. Par exemple, les ligands BAFF et APRIL contrôlent la croissance et la survie des cellules Β matures à différents stades de différenciation. Ces ligands existent souvent sous forme d'homotrimères (3-mer), bien que des héteromères aient été décrits pour la lymphotoxine α1β2 et pour BAFF et APRIL. Dans le cas de BAFF, vingt trimères peuvent, telle une particule virale, s'assembler en 60-mer qui surpasse le 3-mer pour signaler dans des cellules Β primaires. Un crible effectué dans des cellules 293T, par co-transfection de ligands différemment marqués, a permis d'identifier six nouveaux heteromères dont la spécificité n'a, hélas, pas correspondu à celle d'un récepteur orphelin de la famille du TNFR. Les connaissances sur la spécificité de liaison aux récepteurs et la capacité à signaler des heteromères de BAFF et d'APRIL sont fragmentaires. Une méthode pour produire et purifier des heteromères "simple chaîne" de stoechiométrie déterminée a été mise au point, et les réactifs ainsi obtenus utilisés pour démontrer que BAFF2APRIL, comme BAFF, lie tous les récepteurs de BAFF - c'est-à-dire BAFFR, TACI et BCMA -, alors qu'APRIL2BAFF et APRIL ne lient que TACI et BCMA. Les héteromères peuvent transmettre des signaux, parfois aussi bien et parfois plus faiblement que les homomères, selon les récepteurs. Un système prometteur pour mesurer l'activité des ligands simple chaîne in vivo a été mis au point. Il mesure la réapparition de cellules Β matures dans des souris déficientes pour BAFF après administration des ligands. Pour s'attaquer à la question de l'importance physiologique du 60-mer de BAFF, ime mutation empêchant l'assemblage en 60-mer sans affecter la capacité à former des trimères a été identifiée. Cette mutation (E247K) a été introduite par recombinaison homologue dans des cellules souches embryonnaires de souris qui sont utilisées pour obtenir des souris déficientes en BAFF 60-mer. Les résultats de ces travaux contribueront à mieux cerner le rôle des différentes formes de BAFF et d'APRIL produites en excès dans plusieurs maladies auto-immunes.

Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands.

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL.

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.

Enforced covalent trimerisation of soluble feline CD134 (OX40)-ligand generates a functional antagonist of feline immunodeficiency virus.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumour necrosis factor receptor superfamily (TNFRSF) and all primary viral strains tested to date use CD134 for infection. To investigate the effect of the natural ligand for CD134 on FIV infection, feline CD134L was cloned and expressed in soluble forms. However, in contrast to murine or human CD134L, soluble feline CD134L (sCD134L) did not bind to CD134. Receptor-binding activity was restored by enforced covalent trimerisation following the introduction of a synthetic trimerisation domain from tenascin (TNC). Feline and human TNC-CD134Ls retained the species-specificity of the membrane-bound forms of the ligand while murine TNC-CD134L displayed promiscuous binding to feline, human or murine CD134. Feline and murine TNC-CD134Ls were antagonists of FIV infection; however, potency was both strain-specific and substrate-dependent, indicating that the modulatory effects of endogenous sCD134L, or exogenous CD134Lbased therapeutics, may vary depending on the viral strain.

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.