Julie Dam

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One‐third of the ∼400 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 MT1 and MT2 melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT2 did not modify MT2 function, GPR50 abolished high‐affinity agonist binding and G protein coupling to the MT1 protomer engaged in the heterodimer. Deletion of the large C‐terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT1. 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.

Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

G‐protein‐coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand‐independent functions for these receptors. This emerging concept is connected with the observation that orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by so doing regulate the function of the latter. Pairing orphan GPCRs with their potential heterodimerization partners will have a major impact on our understanding of the extraordinary diversity offered by GPCR heterodimerization and, in addition, will constitute a novel strategy to elucidate the function of orphan receptors that needs to be added to the repertoire of ‘deorphanization’ strategies.

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

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.

A role for the melatonin-related receptor GPR50 in leptin signaling, adaptive thermogenesis, and torpor.

The ability of mammals to maintain a constant body temperature has proven to be a profound evolutionary advantage, allowing members of this class to thrive in most environments on earth. Intriguingly, some mammals employ bouts of deep hypothermia (torpor) to cope with reduced food supply and harsh climates [1, 2]. During torpor, physiological processes such as respiration, cardiac function, and metabolic rate are severely depressed, yet the neural mechanisms that regulate torpor remain unclear [3]. Hypothalamic responses to energy signals, such as leptin, influence the expression of torpor [4-7]. We show that the orphan receptor GPR50 plays an important role in adaptive thermogenesis and torpor. Unlike wild-type mice, Gpr50(-/-) mice readily enter torpor in response to fasting and 2-deoxyglucose administration. Decreased thermogenesis in Gpr50(-/-) mice is not due to a deficit in brown adipose tissue, the principal site of nonshivering thermogenesis in mice [8]. GPR50 is highly expressed in the hypothalamus of several species, including man [9, 10]. In line with this, altered thermoregulation in Gpr50(-/-) mice is associated with attenuated responses to leptin and a suppression of thyrotropin-releasing hormone. Thus, our findings identify hypothalamic circuits involved in torpor and reveal GPR50 to be a novel component of adaptive thermogenesis in mammals.

Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway.

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.

Increased expression of Fibroblast Growth Factor 21 (FGF21) during chronic undernutrition causes Growth Hormone insensitivity in chondrocytes by inducing leptin receptor overlapping transcript (LEPROT) and leptin receptor overlapping transcript-like 1 (LEPROTL1) expression

Background: FGF21 causes GH insensitivity. Results: Increased FGF21 expression induces LEPROT and LEPROTL1 expression. Inhibition of LEPROT or LEPROTL1 in growth plate chondrocytes prevents the FGF21-mediated inhibition of the GH stimulatory effects on chondrocyte function and IGF-1 expression. Conclusion: FGF21 prevents the GH effects on chondrocytes by activating LEPROT and LEPROTL1. Significance: LEPROT and LEPROTL1 mediate the FGF21 inhibition of GH action. During calorie restriction in mice, increased expression of FGF21 causes growth attenuation and growth hormone (GH) insensitivity. Previous evidence also indicates that fasting-associated increased expression of leptin receptor overlapping transcript (LEPROT) and LEPROT-like 1 (LEPROTL1) (two proteins that regulate intracellular protein trafficking) reduces GH receptor cell-surface expression in the liver. Thus, we hypothesized that FGF21 causes GH insensitivity through regulation of LEPROT and/or LEPROTL1 expression. After 4 weeks of food restriction, LEPROT and LEPROTL1 mRNA expression in the liver and in the tibial growth plate of wild-type (WT) mice was increased compared with WT mice fed ad libitum. In Fgf21 knock-out (KO) mice, LEPROT and LEPROTL1 mRNA expression in food-restricted and fed ad libitum was similar, with the exception of a subgroup of food-restricted Fgf21 KO mice treated with recombinant human (rh) FGF21 that experienced increased LEPROT and LEPROTL1 mRNA expression compared with untreated food-restricted Fgf21 KO mice. In cultured growth plate chondrocytes, FGF21 stimulated LEPROT and LEPROTL1 mRNA expression, with such effect being prevented in chondrocytes transfected with FGFR1 siRNA or ERK1 siRNA. In cells transfected with control siRNA, GH increased [3H]thymidine incorporation, collagen X, and IGF-1 mRNA expression, with all effects being prevented by rhFGF21. In addition, rhFGF21 decreased 125I-GH binding. In LEPROT siRNA- and/or LEPROTL1 siRNA-transfected cells, rhFGF21 did not prevent the GH stimulatory effects on thymidine incorporation, collagen X, and IGF-1 expression; furthermore, rhFGF21 did not prevent 125I-GH binding. Consistent with the effects of rhFGF21, LEPROT overexpression in chondrocytes resulted in the inhibition of GH action. Our findings indicate that the increased expression of FGF21 during chronic undernutrition inhibits GH action on chondrocytes by activating LEPROT and LEPROTL1.

Homozygous deletion of an 80kb region comprising part of DNAJC6 and LEPR genes on chromosome 1P31.3 is associated with early onset obesity, mental retardation and epilepsy.

CONTEXT The genomic organization of the LEPR gene is complex and generates three independent transcripts whose respective functions are still poorly understood. METHODS/RESULTS We describe here a 7-year old patient with a homozygous 80 kb deletion in the chromosomal 1p31.3 region with early onset obesity, mental retardation and epilepsy. The deleted region comprises the proximal promoter and exons 1 and 2 of the LEPR gene and exons 5 to 19 of the DNAJC6 gene. The deletion leads to the deficiency of all canonical OB-R isoforms but maintains the B219 OB-R short isoforms controlled by the preserved second LEPR promoter. The DNAJC6 gene encodes auxilin-1, a protein required for clathrin-dependent recycling of synaptic vesicles in neurons that is possibly at the origin of the mental retardation and epilepsy phenotype. The obese phenotype and the absence of signaling-competent OB-R are consistent with previously reported individuals with OB-R deficiency. The deletion eliminates an additional transcript of the LEPR gene that encodes endospanin-1, a protein that has been genetically and biochemically linked to OB-R function. CONCLUSIONS Our study confirms the phenotype of individuals with OB-R deficiency and postulates the effects of auxilin-1 deficiency (mental retardation/epilepsy) and endospanin-1 deficiency (OB-R specific functions) in humans.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.

Hunting for the functions of short leptin receptor isoforms

Alternate splicing of the leptin receptor gene generates six receptor isoforms (ObRa-f) that share a common extracellular and transmembrane domain, but differ in their intracellular segment. The long ObRb isoform has the longest intracellular domain with 302 amino acids and is the only isoform containing functional JAK2 and STAT binding sites, which are essential for signal transduction and transmission of leptin function. Despite this prominent role of ObRb in mediating the function of leptin, its expression levels are much lower and its tissue distribution much more restricted than those of the other ObR isoforms. Thus, due to the heavy interest in ObRb signaling to elucidate the role of leptin, little research has examined the functional roles of the other, much more abundant, ObR isoforms, including the ObRa isoform. In this issue of “Molecular Metabolism”, Li and colleagues address the functionality of the ObRa isoform by engineering an ObRa knockout (KO) mouse model through deletion of an ObRa-specific exon [1]. The authors first investigated the metabolic phenotype of these mice, and report decreased fasting blood glucose and an improved glucose tolerance during chow feeding without changes in body weight and food intake. However, they observed a slight increase of fat mass and body weight during high fat feeding associated with modest leptin resistance and a small decrease in leptin transport into the cerebrospinal fluid. Overall, the ObRa spliced isoform seems to mediate some of leptins effects, but compared to ObRb, its role in the control of metabolism appears to be limited. This result seems to be disappointing at the first glance but appears full of sense when taking into account relative expression levels of ObRa and ObRb in the hypothalamus, the major site of leptin action in regulating energy and glucose homeostasis. Indeed, in addition to the phenotypic characterization of ObRa KO mice, the authors also provide for the first time a comprehensive relative expression profile of the different ObR isoforms in central and peripheral tissues. According to their analyses, ObRa mRNA expression levels in the hypothalamus are very low relative to the two major isoforms in this tissue, ObRb and ObRc, which are at least 10 times more abundant than ObRa. Additionally, similar expression levels of ObRb and ObRc are observed in WT and ObRa knockout mice excluding any confounding compensatory effects. Thus, the marginal expression of the ObRa isoform in the hypothalamus is likely to explain the modest changes in energy and glucose homeostasis in ObRa KO mice under chow diet conditions. Challenging ObRa KO mice with a high fat diet revealed slightly increased fat mass and body weight. In agreement with the very low expression of ObRa in the hypothalamus, leptin responsiveness of the ARC was unchanged in HFD-fed ObRa KO mice. Previous reports have established that the ObRa isoform participates in the transport of leptin into the CNS through the blood brain barrier and that defective transport participates in the establishment and maintenance of the state of leptin resistance [2]. The decrease of leptin transport into the cerebrospinal fluid observed in HFD-fed ObRa KO mice supports this hypothesis. Transport of leptin into the brain may occur through the choroid plexus (CP), blood micro-vessels (V) and circumventricular organs, such as the median eminence, which lack a typical blood brain barrier [3]. Determination of the mRNA levels of the different ObR isoforms in CP and V show that these structures express abundant levels of ObRa, c and e, and to a lesser extent, also ObRb. Based on this observation and that ObRa is thought to be implicated in the transport of leptin, a more obese phenotype would have been expected in high fat-fed mice. The fact that this was not the case might be explained by the substantial up-regulation of ObRb and ObRc isoforms in ObRa KO mice. This compensatory effect renders conclusions concerning the role of ObRa in leptin transport difficult. Furthermore, relative ObR isoform expression levels and possible compensatory effects in ObRa KO mice in the median eminence are currently unknown. This structure is of particular interest due to its close proximity to the hypothalamic arcuate nucleus. Several studies indicate that peripheral tissues also contribute to the effect of leptin on energy and glucose homeostasis [4]. Unfortunately, the ObRa KO model is unable to provide any new insights in this area because the authors show that ablation of the ObRa isoform is largely compensated by the up-regulation of ObRb and ObRc in these tissues leading to similar levels of expression of total ObR. The present work reveals an intriguing compensatory mechanism, which most likely takes place on the level of mRNA splicing generating the different ObR isoforms. Notably, the absence of ObRa in tissues typically expressing significant quantities of this isoform is systematically replaced by ObRb and ObRc isoforms. The biological need to maintain the total ObR expression levels constant is currently not clear. Some tissues, such as the hypothalamus, however, displayed no compensatory increases in other ObR isoforms. This lack of compensation in the hypothalamus could be accounted by the fact that ObRa is expressed at a very low level in the hypothalamus (around 2–3% of all OB-R isoforms) compared to the testis and muscle where OB-Ra mRNA is highly present (around 30%). Increased expression of ObRc in the ObRa KO mice may compensate the functional effects of ObRa deletion since the amino-acid sequence of both isoforms differ by only few amino acids at the C-terminal end (…RTDLV for ObRa versus …KVTV for ObRc). This leads to the interesting question whether ObRa and ObRc have partially redundant functions. One way to answer this question would be the generation of double KO mice of these two most abundantly expressed OB-R isoforms. The study of Li and colleagues clearly places the ObRc isoform in the focus of future work on the leptin receptor. Overall, ObRc is either as abundant (CP, spleen, testis, muscle, small intestine, heart, stomach) or more prevalent than ObRa (cortex, hippocampus, hypothalamus, cerebellum and lung), suggesting that ObRc could be at least as important as ObRa in mediating the functions of leptin. Previous studies had also brought to light a similar or higher expression of ObRc over ObRa [5,6]. Heteromerization between different ObR isoforms has been reported previously [7,8]. Although not shown directly in tissues, engagement of ObRb into heteromers with short ObR isoforms, which are typically expressed in large excess over ObRb, is likely to occur. This would imply that the short isoforms, by forming heteromers with ObRb, could either influence the signaling of the latter or produce a new entity, the heteromer, with its own specific signaling pathways. This option has to be kept in mind for future studies on the role of ObRb versus the short ObR isoforms. The authors generated a mouse model that would help elucidating the role of ObRa in other biological functions of leptin apart from energy homeostasis. Leptin signaling has been proposed to mediate the regulation of respiration, reproduction, inflammation, immune functions, angiogenesis, bone homeostasis, neuronal plasticity, breast and prostate cancer [9,10]. Ob-Ra and Ob-Rc mRNA are highly abundant in the choroid plexus, the brain microvessels, the lung, spleen, testis, muscle, small intestine, kidney, heart and stomach suggesting their participation in leptin function in those tissues. Recently, a role for ObRa in inflammation has been suggested [11]. The pioneering work of Li et al. opens new perspectives in our understanding of the short ObR isoforms. This work corroborates the involvement of ObRa in leptin transport into the brain and suggests that, compared to ObRb, ObRa has minor effect on energy metabolism. Furthermore, it clearly highlights the potential importance of the ObRc isoform and revealed an unexpected autoregulatory mechanism of the overall amount of expressed ObR in a given tissue. Future studies will have to address the different physiological functions of the short ObR isoforms based on these results.

Anti-obesity Phenotypic Screening Looking to Increase OBR Cell Surface Expression

The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.