One-third of the B400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT 1 and MT 2 melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT 2 did not modify MT 2 function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT 1 protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT 1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT 1 . Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo-and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.Membrane proteins such as tyrosine kinase, cytokine, or transforming growth factor receptors have been known for many years to form oligomers, and the link between their oligomerization and activity states has been well established (1). In contrast, G protein-coupled receptor (GPCR) 1 oligomerization has been documented only recently (2), and the relation between receptor activation and oligomerization is still poorly understood. Even the proportion between monomeric and oligomeric receptor species and the exact oligomerization state (dimer, trimer, tetramer, etc.) remains a matter of controversy. Currently, two models are proposed. In the first model GPCR are monomeric in their inactive state, and agonist activation induces the formation of receptor oligomers. This model is based on low basal and strong agonist-induced energy transfer signals observed in fluorescence and bioluminescence resonance energy transfer (FRET, BRET) experiments for the gonadotropin-releasing hormone (3), the somatostatin SSTR5 (4), and the SSTR5/dopamin D2R receptor oligomers (5). The second model proposes that GPCR are constitutively oligomerized and is supported by studies reporting high basal BRET or FRET signals for ␣-mating factor (6), ␤2-adrenergic (␤2AR) (7), tyrothropin-releasing hormone (8), ␦-opioid (9), type A cholecystokinin (10), and dopamine D2 receptors (11). Agonist-promoted increases in signals were observed in some of thes...

The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been described using different substrates and energy donor/acceptor couples. Today, BRET is considered as one of the most versatile techniques for studying the dynamics of protein-protein interactions in living cells. Various studies have applied BRET-based assays to screen new receptor ligands and inhibitors of disease-related-proteases. Inhibitors of protein-protein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET-based methodology, more recent variants, and potential applications to drug screening.

Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals. Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown. Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that ϳ60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin. No further increase in leptin receptor dimerization was detected in the presence of leptin. Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands. Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent. Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization.

Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this ''leptin resistance'' is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.leptin receptor overlapping transcript ͉ leptin resistance ͉ gene therapy ͉ receptor trafficking ͉ metabolic syndrome L eptin and its receptor (OB-R) were initially identified and characterized because of their involvement in the regulation of energy balance, metabolism, and neuroendocrine responses to food intake (1). Subsequently, leptin has also been shown to be important in wound healing (2), angiogenesis (3), and bone mass and immune system regulation (4, 5). OB-R belongs to the class I cytokine receptor family, which typically uses the JAK/STAT signaling pathway (6). The human OB-R gene on chromosome 1 generates multiple transcripts. Several of these transcripts encode at least five OB-R isoforms, including the short OB-Ra isoform and the long signaling-competent OB-Rb isoform (1). An additional transcript is generated from the same locus encoding a protein called OB-RGRP [or leptin receptor overlapping transcript (LEPROT)], which does not share any sequence similarity with OB-R (7). In situ hybridization experiments show coexpression of the OB-R transcript and the associated OB-RGRP transcript in the mouse brain, including hypothalamic regions involved in body weight regulation (8). Evolutionarily conserved coexpression of two ORFs is often observed in prokaryotes and viruses; however, there are few known cases in eukaryotes (9). Mammals have a single OB-RGRP homologue called LEPROTL1 (leptin receptor overlapping transcript-like 1), that has 70% amino acid sequence similarity with OB-RGRP and whose gene maps on chromosome 8 in humans (10). In yeast, Vps55p, a functional homologue of OB-RGRP, plays a role in protein transport from the Golgi to the vacuole and in the late endoc...

Type II-secreted phospholipase A 2 (type II-sPLA 2 ) is expressed in smooth muscle cells during atherosclerosis or in response to interleukin-1␤. The present study shows that the induction of type II-sPLA 2 gene by interleukin-1␤ requires activation of the NFB pathway and cytosolic PLA 2 /PPAR␥ pathway, which are both necessary to achieve the transcriptional process. Interleukin-1␤ induced type II-sPLA 2 gene dose-and time-dependently and increased the binding of NFB to a specific site of type II-sPLA 2 promoter. This effect was abolished by proteinase inhibitors that block the proteasome machinery and NFB nuclear translocation. Type II-sPLA 2 induction was also obtained by free arachidonic acid and was blocked by either AACOCF 3 , a specific cytosolic-PLA 2 inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor which prevents cytosolic PLA 2 activation, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, but not by the cyclooxygenase inhibitor indomethacin, suggesting a role for a lipoxygenase product. Type II-sPLA 2 induction was obtained after treatment of the cells by 15-deoxy-⌬ 12,14 -dehydroprostaglandin J 2 , carbaprostacyclin, and 9-hydroxyoctadecadienoic acid, which are ligands of peroxisome proliferator-activated receptor (PPAR) ␥, whereas PPAR␣ ligands were ineffective. Interleukin-1␤ as well as PPAR␥-ligands stimulated the activity of a reporter gene containing PPAR␥-binding sites in its promoter. Binding of both NFB and PPAR␥ to their promoter is required to stimulate the transcriptional process since inhibitors of each class block interleukin-1␤-induced type II-sPLA 2 gene activation. We therefore suggest that NFB and PPAR␥ cooperate at the enhanceosome-coactivator level to turn on transcription of the proinflammatory type II-sPLA 2 gene.

Scope: Several studies have linked the high intake of lycopene or tomatoes products with lower risk for metabolic diseases. The aim of the present study was to evaluate and to compare the effect of lycopene and tomato powder on obesity associated disorders. Methods and results:Male C57BL/J6 mice were assigned into 4 groups to receive: control diet (CD), high fat diet (HFD), high fat diet supplemented with lycopene or with tomato powder (TP) for 12 weeks. In HFD condition, lycopene and TP supplementation significantly reduced adiposity index, organ and relative organ weights, serum triglycerides, free fatty acids, 8-iso-prostaglandin GF2α and improved glucose homeostasis, but did not affect total body weight. Lycopene and TP supplementation prevented HFD-induced hepatosteatosis and hypertrophy of adipocytes. Lycopene and TP decreased HFD-induced pro-inflammatory cytokine mRNA expression in the liver and in the epididymal adipose tissue. The antiinflammatory effect of lycopene and TP was related to a reduction in the phosphorylation levels of IκB, and p65, and resulted in a decrease of inflammatory proteins in adipose tissue Conclusion:These results suggest that lycopene or TP supplementation display similar beneficial health effects that could be particularly relevant in the context of nutritional approaches to fight obesity-associated pathologies.

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.Leptin and the leptin receptor are key proteins in pathways regulating energy balance and body weight in both humans and rodents (1-3). The leptin receptor is a single membrane-spanning protein of the class I cytokine receptor family. The leptin receptor gene is transcribed in a number of different messenger RNAs by alternative promoter usage and mRNA splicing (4). Most of these transcripts are splice variants that encode receptor isoforms with different cytoplasmic tails, which are referred to as OB-Ra, OB-Rc, OB-Rd (short isoforms), and OB-Rb (long isoform). The long splice variant OB-Rb mediates the majority of the effects of leptin, mainly via the JAK-STAT signaling pathway (1, 5, 6). The exact function of the short isoforms is still unclear. Short and long isoforms follow very similar intracellular membrane trafficking pathways (7-9).Recently, we identified a negative regulator of leptin receptor function (10). This protein, originally named leptin receptor gene-related protein, or OB-RGRP, is encoded by a transcript expressed from the leptin receptor gene (11). The OB-RGRP mRNA shares two exons that are untranslated in leptin receptor transcripts and contains two additional exons that are absent in leptin receptor transcripts. This transcript is named LEPROT (leptin receptor overlapping transcript). This overlapping gene structure is conserved in humans and rodents (11,12). We named this protein endospanin-1, due to its topology and intracellular localization. We have shown that endospanin-1 down-regulates leptin signaling by lowering the expression of the receptor at the cell surface and thus the sensitivity of the cell to leptin. Furthermore, the physiological relevance of endospanin-1 as a regulator of OB-R function was evaluated in mice. The expression of a lentivirus-delivered short hairpin RNA (shRNA) directed against endospanin-1 in the arcuate nucleus of the hypothalamus prevented the...