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.

Induced down-regulation of neuropeptide Y-Y1 receptors delays initiation of kindling.

Neuropeptide Y appears to modulate epileptic seizures differentially according to the receptor subtypes involved. In the hippocampus, neuropeptide Y expression and release are enhanced in different models of epileptogenesis. On the contrary, the expression of Y1 receptors is decreased and it has been shown that activation of these receptors has pro-convulsant effects. The aim of our study was to investigate the role of Y1 receptors during hippocampal kindling epileptogenesis using (i) knock-out mice lacking Y1 receptors and (ii) intrahippocampal infusion of Y1 antisense oligodeoxynucleotide in rats. Y1 knock-out mice showed similar susceptibility to seizure induction and presented no difference in kindling development as compared with their control littermates. Conversely, local hippocampal down-regulation of Y1 receptors during the first week of hippocampal kindling, induced by a local infusion of a Y1 antisense oligodeoxynucleotide, significantly increased seizure threshold intensity and decreased afterdischarge duration. A reverse effect was observed during the week following the infusion period, which was confirmed by a significant decrease in the number of hippocampal stimulations necessary to evoke generalized seizures. At the end of this second week, an up-regulation of Y1 receptors was observed in kindled rats infused with the antisense as compared with the mismatch-treated controls. Our results in the rat suggest that the down-regulation of Y1 receptors in the hippocampus participates in the control of the initiation of epileptogenesis. The lack of an effect of the deficiency of Y1 receptors in the control of kindling development in Y1 knock-out mice could be due to compensatory mechanisms.

Kinase signaling cascades that modulate peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in lipid and glucose homeostasis, inflammation and wound healing. In addition to ligand binding, phosphorylation can also regulate PPARs; the biological effects of phosphorylation depend on the stimulus, the kinase, the PPAR isotype, the residue modified, the cell type and the promoter investigated. The study of this dual regulation mode, which allows PPARs to integrate signals conveyed by lipophilic ligands with those coming from the plasma membrane, may ultimately offer new therapeutic strategies.

Dopamine receptors on dispersed bovine anterior pituitary cells

The dopamine antagonist [3H]-domperidone-[3H]-DOM-bound to a single class of high-affinity (Kd = 1.24 +/- 0.14 nM) and saturable receptors on dispersed bovine anterior pituitary (AP) cells. The binding of [3H]-DOM was stereoselective and reversible with agonists and antagonists. Dopamine competitions for [3H]-DOM binding modeled best for a single site consistent with an interaction with a homogeneous population of receptors. The mean number of specific binding sites labeled by [3H]-DOM was 53,000 per cell in dispersed AP cells consisting of 42% lactotrophs. Dispersed bovine AP cells attached to extracellular matrix within 3 h, and prolactin secretion from these cells was effectively inhibited by dopamine. Several observations suggested that [3H]-DOM-labeled receptors on dispersed bovine AP cells were restricted to the outer plasma membrane and not internalized. These included (1) the rapid and complete dissociation of specific [3H]-DOM binding; (2) the ability of treatment with acid or proteolytic enzymes to entirely remove specifically bound [3H]-DOM, and (3) the lack of effect of metabolic inhibitors on specific [3H]-DOM binding.

RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis.

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.

No implication of thromboxane-prostanoid receptors in reactive hyperemia of skin skeletal muscle in human forearm

L'hyperhémie réactive, définie comme l'augmentation transitoire du flux sanguin après une courte période d'ischémie, pourrait être influencée par des vasoconstricteurs de la famille des prostanoïdes, telle que la thromboxane. Le terutroban (S18886) est un antagoniste spécifique des récepteurs à la thromboxane. L'étude présentée a cherché à déterminer l'effet du terutroban sur l'hyperhémie réactive dans la peau et le muscle squelettique de l'avant-bras de volontaires sains. Vingt volontaires sains ont été randomisés en aveugle pour recevoir oralement 30mg/j de terutroban ou un placebo pendant 5 jours puis réciproquement pendant une deuxième période de 5 jours, selon un schéma cross-over. L'ischémie transitoire a été provoquée par l'occlusion de l'artère brachiale par une manchette gonflée au dessus de la pression systolique. L'hyperhémie réactive était évaluée dans les tissus de l'avant- bras, en mesurant le flux sanguin, pour la peau par une méthode laser Doppler, et pour le muscle au moyen d'une pléthysmographie par jauge de contrainte durant une occlusion veineuse. Au premier et au dernier jour de chaque période de traitement, l'hyperhémie réactive était mesurée avant et 2 heures après l'ingestion du comprimé. Que ce soit dans la peau ou le muscle, le terutroban n'a pas montré d'effet sur le flux de pic post-occlusion ni sur la réponse globale d'hyperhémie, exprimée en aire sous la courbe. En conclusion, dans la peau et le muscle de sujets sains, l'hypérémie réactive n'est pas influencée par les récepteurs spécifiques à la thromboxane.

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Role of MyD88 and Toll-like receptors 2 and 4 in the sensing of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88(-/-), TLR4(-/-), and TLR2(-/-) mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

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.

TWEAK can induce cell death via endogenous TNF and TNF receptor 1.

TWEAK is a recently cloned novel member of the TNF ligand family. Here we show that soluble TWEAK is sufficient to induce apoptosis in Kym-1 cells within 18 h. TWEAK-induced apoptosis is indirect and is mediated by the interaction of endogenous TNF and TNF receptor (TNFR)1, as each TNFR1-Fc, neutralizing TNF-specific antibodies and TNFR1-specific Fab fragments efficiently antagonize cell death induction. In addition to this indirect mode of action, co-stimulation of Kym-1 cells with TWEAK enhances TNFR1-mediated cell death induction. In contrast to TNF, TWEAK does only modestly activate NF-kappaB or c-jun N-terminal kinase (JNK) in Kym-1 cells. Although TWEAK binding to Kym-1 cells is easily detectable by flow cytometric analysis, we found neither evidence for expression of the recently identified TWEAK receptor Apo3/TRAMP/wsl/DR3/LARD, nor indications for direct interactions of TWEAK with TNFR. Together, these characteristics of TWEAK-induced signaling in Kym-1 cells argue for the existence of an additional, still undefined non-death domain-containing TWEAK receptor in Kym-1 cells.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

The effects of gamma-hydroxybutyrate, baclofen and GABAB receptors on brain activity and sleep in mice and humans

Currently, there is an increased interest in γ-hydroxybutyric acid (GHB) and its effects onsleep. This compound, sometimes referred to as 'rape drug', was recently approved as atreatment for the sleep disorder narcolepsy. Although several studies suggest that GHBinduces slow-wave sleep duration and improves sleep quality by increasing EEG slow-waveactivity, others question its ability to induce physiological sleep. GHB's mechanism of actionis still unclear, although in vivo and in vitro it seems to act at high doses as a low-affinityagonist of GABAB receptors. Furthermore, the role GABAB receptors play in sleep and theelectroencephalogram (EEG) is largely unknown.The aim of this project was therefore to investigate the effects of GHB on sleep and EEG, theinvolvement of GABAB receptors in mediating these effects, as well as the intrinsic role ofeach GABAB receptor subunit in the regulation of sleep. Thus, we administered GHB andbaclofen (BAC, a high-affinity agonist at GABAB receptor) to mice lacking the different GABABreceptor subunits and to healthy human volunteers.Our results, both in mice and humans, showed that GHB produced slow waves exclusivelythrough the stimulation of GABAB receptors, but did not induce physiological sleepnecessary to reduce sleep need and to increase cognitive performance. Unlike GHB, BACaffected the homeostatic regulation of sleep (sleep need) and induced a delayedhypersomnia. Finally, GABAB receptor and its subunits seem to play an important role insleep and in particular its circadian distribution.

The peroxisome proliferator-activated receptors at the cross-road of diet and hormonal signalling.

The peroxisome proliferator-activated receptors (PPARs) are members of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. To date, three isotypes have been identified, alpha, beta and gamma, encoded by three different genes. The alpha isotype is expressed at high levels in the liver where it has a role in lipid oxidation. Its expression and activity follow a diurnal rhythm that parallels the circulating levels of corticosterone in the bloodstream. The gamma isotype on the other hand, is mainly expressed in adipose tissue and has a critical role in adipocyte differentiation and lipid storage. The function of the ubiquitously expressed isotype, PPAR beta, remains to be determined. Besides fulfilling different roles in lipid metabolism, the different PPAR isotypes also have different ligand specificities. A new approach to identify ligands was developed based on the ligand-dependent interaction of PPAR with the recently characterized co-activator SRC-1. This so-called CARLA assay has allowed the identification of fatty acids and eicosanoids as PPAR ligands. Although the evidence clearly links PPAR isotypes to distinct functions, the molecular basis for this isotype-specificity is still unclear. All three isotypes are able to bind the same consensus response element, formed by a direct repeat of two AGGTCA hexamers separated by one base, though with different affinities. We recently demonstrated that besides the core DR-1 element, the 5' flanking sequence should be included in the definition of a PPRE. Interestingly, the presence of this flanking sequence is of particular importance in the context of PPAR alpha binding. Moreover, it reflects the polarity of the PPAR-RXR heterodimer on DNA, with PPAR binding to the 5' half-site and RXR binding to the 3' half-site. This unusual polarity may confer unique properties to the bound heterodimer with respect to ligand binding and interaction with co-activators and corepressors.