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Ghrelin produced in the stomach promotes food intake and GH secretion, and acts as an anabolic peptide during starvation. Ghrelin binds to the growth hormone secretagogue receptor, a G protein-coupled receptor (GPCR), whose high-resolution complex structures have been determined in the apo state and when bound to an antagonist. Anamorelin, a low-molecular-weight ghrelin agonist, has been launched in Japan for the treatment of cancer cachexia, and its therapeutic potential has attracted attention due to the various biological activities of ghrelin. In 2019, liver-expressed antimicrobial peptide (LEAP2), initially discovered as an antimicrobial peptide produced in the liver, was identified to be upregulated in the stomach of diet-induced obese mice after vertical sleeve gastrectomy. LEAP2 binds to the GHSR and antagonizes ghrelin's activities. The serum concentrations of human LEAP2 are positively correlated with body mass index, body fat accumulation, and fasting serum concentrations of glucose and triglyceride. Serum LEAP2 elevated and ghrelin reduced in obesity. Ghrelin and LEAP2 regulate body weight, food intake, and GH and blood glucose concentrations, and other physiological phenomena through their interactions with the same receptor, GHSR.

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design., (© 2024. The Author(s).)

The µ-opioid receptor (µOR) is a well-established target for analgesia 1 , yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl 2 , a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ida, Tominaga, Iwamoto, Kurogi, Okura, Shimada, Kato, Kuwano, Ode, Nagata, Kitamura, Yazawa, Sato-Hashimoto, Yasuda, Miyazato, Shiimura, Sato and Kojima.)

Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H 4 receptor (H 4 R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H 4 R-G q complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe344 7.39 , which, in turn, form the "aromatic slot". The results provide insights into the molecular underpinnings of the agonism of H 4 R and subtype selectivity of histamine receptors, and show that the H 4 R structures may be valuable in rational drug design of drugs targeting the H 4 R., (© 2023. Springer Nature Limited.)

The regulation of fetal development by bioactive substances such as hormones and neuropeptides derived from the gestational mother is considered to be essential for the development of the fetus. On the other hand, it has been suggested that changes in the physiological state of the pregnant mother due to various factors may alter the secretion of these bioactive substances and induce metabolic changes in the offspring, such as obesity, overeating, and inflammation, thereby affecting postnatal growth and health. However, our knowledge of how gestational maternal bioactive substances modulate offspring physiology remains fragmented and lacks a systematic understanding. In this mini-review, we focus on ghrelin, which regulates growth and energy metabolism, to advance our understanding of the mechanisms by which maternally derived ghrelin regulates the growth and health of the offspring. Understanding the regulation of offspring growth by maternally-derived ghrelin is expected to clarify the fetal onset of metabolic abnormalities and lead to a better understanding of lifelong health in the next generation of offspring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sato, Ida, Shiimura, Matsui, Oishi and Kojima.)

Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In addition to the serotonin 5-HT 2A receptor (5-HT 2A R), the dopamine D 2 receptor (D 2 R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D 2 R have been described in complex with the inverse agonists risperidone (D 2 R ris ) and haloperidol (D 2 R hal ). Here we describe the structure of human D 2 R in complex with spiperone (D 2 R spi ). In D 2 R spi , the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D 2 R ris and D 2 R hal , demonstrating that ECL2 in D 2 R is highly dynamic. Moreover, D 2 R spi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D 2 R and 5-HT 2A R. Together with D 2 R ris and D 2 R hal , the structural information of D 2 R spi should be of value for designing novel antipsychotics with improved safety and efficacy.

Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin's activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation.

Ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), have been found in a variety of malignant tumor tissues, suggesting a biological function of the ghrelin/GHS-R axis in tumor growth and progression. Among central nervous system tumors, primary central nervous system lymphomas (PCNSLs) are relatively rare and characterized by a rapid progression and poor prognosis. In order to clarify ghrelin expression and its functional role in promoting tumor growth and progression in PCNSLs, we undertook an immunohistochemical investigation for ghrelin and GHS-R expression in 43 patients and tested the effect of ghrelin inhibition on lymphoma cells. Furthermore, we investigated the expression of CD105, a marker for tumor angiogenesis, to explore its association with the ghrelin/GHS-R axis. The Kaplan-Meier method and Cox's proportional hazards regression model were used to determine the association of ghrelin/GHS-R expression with overall survival rate. The immunohistochemical study showed moderate/strong immunostaining of cells for ghrelin and GHS-R in 40 patients (93.0%) and 39 patients (90.7%), respectively. A ghrelin inhibitor did not affect tumor cell proliferation in vitro. Expression levels of ghrelin and GHS-R were divided into high and low groups by the rate of moderate-strong staining cells to tumor cells. The survival rate was significantly lower in patients with high GHS-R expression (P = 0.0368 by log-rank test; P = 0.0219 by Wilcoxon test). In addition, multivariate analysis of overall survival using Cox's proportional hazards regression model indicated that GHS-R was a significant independent prognostic factor (P = 0.0426). CD105 expression on tumor vessels was positive in 33 patients (33/37, 89.2%). There was a positive correlation between the moderate-strong staining rate of ghrelin and CD105-positive vessel count. These results indicated that the ghrelin/GHS-R axis plays a potential role in promoting tumor growth and progression through neoangiogenesis, rather than the proliferation of tumor cells., (© 2020 Japanese Society of Neuropathology.)

Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT 1 R) and AngII type-2 receptor (AT 2 R), are central to its mechanism of action. We solved the crystal structure of human AT 2 R bound to AngII and its specific antibody at 3.2-Å resolution. AngII (full agonist) and [Sar 1 , Ile 8 ]-AngII (partial agonist) interact with AT 2 R in a similar fashion, except at the bottom of the AT 2 R ligand-binding pocket. In particular, the residues including Met128 3.36 , which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT 2 R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE 2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT 1 R) and AngII type 2 receptor (AT 2 R). We have solved the crystal structure of human AT 2 R binding the peptide ligand [Sar 1 , Ile 8 ]AngII and its specific antibody at 3.2-Å resolution. [Sar 1 , Ile 8 ]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT 1 R and AT 2 R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.

To examine the gene expression of ghrelin, a growth hormone releasing and appetite stimulating hormone from stomach, we constructed human ghrelin promoter-reporter vectors and analyzed the promoter activity. The ghrelin promoter activity was high when cultured cells that express ghrelin mRNA endogenously like TT or ECC10 cells were used, indicating that these cells contain factors necessary for full expression of the human ghrelin gene. The human ghrelin promoter contains both positive and negative regulatory regions. A transient decrease of the promoter activity was found when the reporter vector with the -1600 fragment of the human ghrelin promoter was transfected into cultured cells. We then examined the effect of several transcription factors on the ghrelin promoter activity and found that NF-κB suppressed and that Nkx2.2, a homeodomain-containing transcription factor that is important for ghrelin cell development in pancreas, activates the promoter activity. These transcription factors may be possible targets for the control of ghrelin gene expression.

Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein coupled receptor. Ghrelin has various physiological functions, such as the stimulation of growth hormone release and of appetite, and fat accumulation. Ghrelin is the only peripheral hormone to transmit satiety signal. Mature ghrelin peptide is consisted of 28 amino acid residues, and is unusual among peptide hormones in that Ser3 is n-octanoylated to obtain. Furthermore, this modification is essential for ghrelin's activity. In order to add this side chain to acyl ghrelin, it is necessary for the recently discovered enzyme, ghrelin-O-acyl transferase (GOAT). Therefore, to understand of ghrelin's functions, it is useful to obtain the knowledge on structures and functions of ghrelin, ghrelin receptor and GOAT. Here, we review our current understanding of the structures and functions of ghrelin, and the relation between obesity and ghrelin. Finally, we referred to the ghrelin and related substances as a drug design target for obesity., (Copyright © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.)

Cold exposure and β3-adrenergic receptor agonist (CL316,243) treatment induce the production of beige cells, which express brown adipocytes(BA)-specific UCP1 protein, in white adipose tissue (WAT). It remains unclear whether the beige cells, which have different gene expression patterns from BA, express BA-characteristic fatty acid oxidation (FAO) proteins. Here we found that 5 day cold exposure and CL316,243 treatment of WAT, but not CL316,243 treatment of primary adipocytes of C57BL/6J mice, increased mRNA levels of BA-characteristic FAO proteins. These results suggest that BA-characteristic FAO proteins are induced in beige cells in a different pathway from UCP1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein-coupled receptor. Ghrelin is a 28-amino acid peptide existing in two major forms: n-octanoyl-modified ghrelin, which possesses an n-octanoyl modification on serine-3 and des-acyl ghrelin. Fatty acid modification of ghrelin is essential for ghrelin-induced growth hormone release from the pituitary and appetite stimulation. This acyl-modification of ghrelin is catalysed by ghrelin-O-acyl transferase recently identified. Despite the number of innovative advancements in this field of research, there are still many aspects of ghrelin function and biosynthesis process that remain to be clarified. Here, we review the current understanding of the structure, regulation and function of ghrelin; this review is intended for researchers who will be involved in this field in the future.

The I-mfa domain proteins I-mfa and HIC are considered to be candidate tumor suppressor genes and have been shown to be involved in transcriptional regulation. We show here that I-mfa and HIC specifically interact with SEI-1 through their C-terminal I-mfa domains in vivo. This interaction affects the intracellular localization of I-mfa and requires the region of SEI-1 between 30 and 90 amino acids, which includes its SERTA domain, and results in repression of its intrinsic transcriptional activity. I-mfa also decreases the levels of the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA, the expression of which is coregulated by E2F·DP-1 and SEI-1 in an interaction-dependent manner in vitro. In addition, I-mfa also specifically interacts with other SERTA domain-containing proteins, including SEI-2, SEI-3, SERTAD3 and SERTAD4, through its I-mfa domain in vivo. This interaction also affects the intracellular localization of I-mfa and represses the intrinsic transcriptional activities of SEI-2 and SERTAD3, which are also involved in the E2F-dependent transcription. These data reveal for the first time that I-mfa domain proteins interact with SERTA domain proteins and negatively regulate their transcriptional activity. Because SEI-1, SEI-2 and SERTAD3, whose intrinsic transcriptional activities are repressed by I-mfa, are suggested to be oncogenes, I-mfa domain proteins may be involved in their oncogenic functions by negatively regulating their transcriptional activities., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)