Expression and intracellular localization of leptin receptor long isoform-GFP chimera

Lundin, Anna; Rondahl, Helena; Walum, Erik; Wilcke, Mona

doi:10.1016/s0167-4889(00)00114-2

Cited by 25 publications

(24 citation statements)

References 37 publications

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“…2B), confirming the predominant localization of OB-R in intracellular compartments. A similar localization has previously been reported for another OB-R l -GFP fusion construct (30).…”

Section: Functional Expression Of Ob-r Fusion Proteins-c-terminalsupporting

confidence: 88%

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Couturier¹,

Jockers

2003

Journal of Biological Chemistry

116

113

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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.

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“…2B), confirming the predominant localization of OB-R in intracellular compartments. A similar localization has previously been reported for another OB-R l -GFP fusion construct (30).…”

Section: Functional Expression Of Ob-r Fusion Proteins-c-terminalsupporting

confidence: 88%

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Couturier¹,

Jockers

2003

Journal of Biological Chemistry

116

113

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“…We recently showed that OB-R internalizes via a clathrin-mediated pathway and is transported to lysosomes where it is degraded. Most OB-Rs (endogenously expressed or transfected) are localized in intracellular membranes (16)(17)(18)(19)(20)(21)28). Importantly, these receptors are fully functional in terms of ligand binding (19,20).…”

Section: Regulation Of Ob-r Trafficking By Ob-rgrpmentioning

confidence: 99%

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

Couturier

Sarkis

Séron

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

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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...

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“…This suggests that OB-Rs turn over rapidly, and/or that the levels of OB-R cell surface expression are subject to an unusual kind of regulation. In vitro studies with transfected leptin receptors have confirmed the existence of a large intracellular OB-R pool (10,19,20). The functional meaning of this distribution, and the relationship between the intracellular pools of receptor and the cell surface, where the interaction with leptin occurs, have not yet been investigated, and very little is known about its intracellular traffic.…”

mentioning

confidence: 99%

Low Levels of Expression of Leptin Receptor at the Cell Surface Result from Constitutive Endocytosis and Intracellular Retention in the Biosynthetic Pathway

Belouzard

Delcroix

Rouillé

2004

Journal of Biological Chemistry

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The leptin receptor is mainly localized in intracellular compartments in target tissues. To study the mechanisms leading to this intracellular localization, two main isoforms of leptin receptors, OB-Ra and OB-Rb, were expressed in HeLa cells. Both isoforms were localized at steady state in the trans-Golgi network, in endosomes, and to a lesser extent, at the cell surface. They turned over with a half-life of less than 2 h. Both isoforms of leptin receptors were constitutively endocytosed in a ligandindependent manner and degraded in lysosomes with no evidence of recycling to the cell surface or to the transGolgi network. The endocytosis was inhibited by the deletion of the cytoplasmic domain. Newly synthesized leptin receptors were partially retained in the Golgi complex or in a post-Golgi intracellular compartment. The transmembrane domain was found to be important for this intracellular retention in the biosynthetic pathway, whereas the cytoplasmic domain was not involved. The data suggest that the low levels of expression of leptin receptors at the cell surface results from partial retention in the biosynthetic pathway, coupled to constitutive removal from the plasma membrane via ligand-independent, constitutive endocytosis.Leptin is a polypeptide hormone secreted by adipocytes, which plays a major role in the regulation of food intake, energy expenditure, and neuroendocrine functions (1, 2). Leptin action is mediated by receptors that are located in the hypothalamus and in many peripheral tissues. Leptin receptors (OB-R) 1 are type I membrane glycoproteins structurally related to class I cytokine receptors (3). Different OB-R isoforms have been described, including OB-Ra and OB-Rb. OB-Rb is often referred to as the long isoform of the leptin receptor. OB-Ra, a short isoform, is the major form of leptin receptor found in most peripheral tissues. Both OB-Ra and OB-Rb are co-expressed in a large number of tissues.All known OB-R isoforms originate from a single gene by alternative splicing of the transcript and only differ in their C termini. OB-Ra and OB-Rb share identical N-terminal leptin binding domains, single spanning transmembrane (TM) domains, and the 29 proximal residues of their cytoplasmic domains. They diverge in the C-terminal part of their cytoplasmic domains. OB-Ra has a unique 5-residue long C-terminal extension, which is changed in OB-Rb for a 273-residue long extension (3). OB-Rb has been shown to activate various signal transduction pathways. The best characterized leptin-induced transduction pathway triggered by OB-Rb is Janus tyrosine kinase 2 (JAK2)-mediated activation of signal transducer and activator of transcription 3 (STAT3) (4, 5). OB-Rb was also found to activate the phosphoinositide 3-kinase (6), mitogen-activated protein kinase (7), and 5Ј-AMP-activated protein kinase (8) pathways in response to leptin. In contrast, the function of OB-Ra remains undefined. It has been shown that OB-Ra could bind to and activate JAK2 in vitro (9, 10), but there is no evidence that OB-Ra actually has...

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Expression and intracellular localization of leptin receptor long isoform-GFP chimera

Cited by 25 publications

References 37 publications

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

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

Low Levels of Expression of Leptin Receptor at the Cell Surface Result from Constitutive Endocytosis and Intracellular Retention in the Biosynthetic Pathway

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