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In the developing hypothalamus, the fat-derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lep ) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lep mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin-stimulated growth, we treated Lep mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti-Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9- or 11-day treatments found to be more effective than shorter (5-day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lep females, but only to the DMHv in Lep males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin-dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.
© 2017 Wiley Periodicals, Inc.
The human Y receptor (YR) and its cognate ligand, pancreatic polypeptide (PP), are involved in the regulation of energy expenditure, satiety, and food intake. This system represents a potential target for the treatment of metabolic diseases and has been extensively investigated and validated in vivo. Here, we present the compound tBPC (tert-butylphenoxycyclohexanol), a novel and selective YR positive allosteric modulator that potentiates YR activation in G-protein signaling and arrestin3 recruitment experiments. The compound has no effect on the binding of the orthosteric ligands, implying its allosteric mode of action at the YR and evidence for a purely efficacy-driven positive allosteric modulation. Finally, the ability of tBPC to selectively potentiate YR agonism initiated by PP was confirmed in mouse descending colon mucosa preparations expressing native YR, demonstrating YR positive allosteric modulation in vitro.
The neuropeptide Y receptor (Y R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
The human neuropeptide Y receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hYR desensitization and endocytosis. The role of distinct segments of the hYR carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hYR and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif EESEHLPLSTVHTEVSKGS was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y receptor, which regulates arrestin-3 recruitment and receptor endocytosis.
Copyright Â© 2016 Elsevier Inc. All rights reserved.
The human neuropeptide Y4 receptor (Y4R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y4R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y4R activity. Compounds were characterized for their potency and their effects at the human Y4R and as well as their selectivity towards Y1R, Y2R and Y5R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y4R.
Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Hyperarousal and sleep disturbances are common, debilitating symptoms of post-traumatic stress disorder (PTSD). PTSD patients also exhibit abnormalities in quantitative electroencephalography (qEEG) power spectra during wake as well as rapid eye movement (REM) and non-REM (NREM) sleep. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for PTSD, provide modest remediation of the hyperarousal symptoms in PTSD patients, but have little to no effect on the sleep-wake architecture deficits. Development of novel therapeutics for these sleep-wake architecture deficits is limited by a lack of relevant animal models. Thus, the present study investigated whether single prolonged stress (SPS), a rodent model of traumatic stress, induces PTSD-like sleep-wake and qEEG spectral power abnormalities that correlate with changes in central serotonin (5-HT) and neuropeptide Y (NPY) signaling in rats. Rats were implanted with telemetric recording devices to continuously measure EEG before and after SPS treatment. A second cohort of rats was used to measure SPS-induced changes in plasma corticosterone, 5-HT utilization, and NPY expression in brain regions that comprise the neural fear circuitry. SPS caused sustained dysregulation of NREM and REM sleep, accompanied by state-dependent alterations in qEEG power spectra indicative of cortical hyperarousal. These changes corresponded with acute induction of the corticosterone receptor co-chaperone FK506-binding protein 51 and delayed reductions in 5-HT utilization and NPY expression in the amygdala. SPS represents a preclinical model of PTSD-related sleep-wake and qEEG disturbances with underlying alterations in neurotransmitter systems known to modulate both sleep-wake architecture and the neural fear circuitry.
Pancreatic polypeptide (PP) is a satiety-inducing gut hormone targeting predominantly the Y4 receptor within the neuropeptide Y multiligand/multireceptor family. Palmitoylated PP-based ligands have already been reported to exert prolonged satiety-inducing effects in animal models. Here, we suggest that other lipidation sites and different fatty acid chain lengths may affect receptor selectivity and metabolic stability. Activity tests revealed significantly enhanced potency of long fatty acid conjugates on all four Y receptors with a preference of position 22 over 30 at Y1 , Y2 and Y5 receptors. Improved Y receptor selectivity was observed for two short fatty acid analogues. Moreover, [K(30)(E-Prop)]hPP2-36 (15) displayed enhanced stability in blood plasma and liver homogenates. Thus, short chain lipidation of hPP at key residue 30 is a promising approach for anti-obesity therapy because of maintained selectivity and a sixfold increased plasma half-life.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.
Copyright © 2014 Elsevier Inc. All rights reserved.
Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr(2.64) interacts with Tyr(27) of hPP through hydrophobic contacts. Asn(7.32) is affected by modifications on position Arg(33) of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe(7.35) is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6-TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.