ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Surface translocation of Kir2.1 channel induces IL-1ß secretion in microglia.

One of the major properties of microglia is to secrete cytokines as a reaction to stress such as lipopolysaccharide (LPS) application. The mechanism of cytokine secretion from the microglia upon stress through the inflammasome-mediated release process is well studied, and the voltage-gated Kv1.3 channel is known to play an important role in this process. Most previous studies investigated long-term inflammasome-mediated cytokine release (at least over 4 h) and there are only a few studies on the acute reaction (within minutes order) of the microglia to stress and its cytokine secretion capacity. In this study, we found that LPS induced an increase in Kir2.1 current within 15 min after administration but had no effect on voltage-dependent outward currents. Moreover, cytological and western blot analysis revealed that the increase in the Kir2.1 channel current after LPS administration was induced by the translocation of Kir2.1 from the cytoplasm to the cell surface. From an experiment using the inhibitor and trafficking mutation of Kir2.1, an increase in Kir2.1 was found to contribute to the secretion of the inflammatory cytokine, IL-1ß. Although the physiological significance of this acute IL-1ß secretion remains unclear, our present data imply that Kir2.1 translocation functions as a regulator of IL-1ß secretion, and therefore becomes a potential target to control cytokine release from microglia.

Soluble interleukin-2 receptor as a predictive and prognostic marker for patients with familial breast cancer.

The incidence of breast cancer increases annually, and it has become common within families of breast cancer patients. Interleukin-2 activates cytotoxic T lymphocytes, which are important for cancer immunity. To identify markers of increased familial breast cancer risk, soluble interleukin-2 receptor levels and immunologic factors were investigated in familial breast cancer and non-familial breast cancer patients. Of 106 untreated breast cancer patients in this study, 24 had familial breast cancer and 82 had non-familial breast cancer. The patients' soluble interleukin-2 receptor, interleukin-10, vascular endothelial growth factor, interleukin-17, regulatory T cell, myeloid-derived suppressor cell, white blood cell, and C-reactive protein levels, and their neutrophil-to-lymphocyte ratios were measured, and their prognoses were compared according to the soluble interleukin-2 receptor levels. Additionally, postoperative tissues from the patients with high soluble interleukin-2 receptor levels were stained with programmed cell death ligand 1 and cluster of differentiation 8. The soluble interleukin-2 receptor level in the familial breast cancer patients was significantly higher, and it showed significantly stronger correlations with the neutrophil-to-lymphocyte ratio and the interleukin-10, vascular endothelial growth factor, interleukin-17, regulatory T cell, myeloid-derived suppressor cell, white blood cell, and C-reactive protein levels, than in the non-familial breast cancer patients. The regulatory T cell and myeloid-derived suppressor cell levels were significantly higher in the patients with high soluble interleukin-2 receptor levels, and the overall survival and disease-free-survival rates were significantly worse for the familial breast cancer patients than for the non-familial breast cancer patients. Triple-negative breast cancer tissues from the familial breast cancer patients with high soluble interleukin-2 receptor levels stained well for programmed cell death ligand 1 and cluster of differentiation 8. Soluble interleukin-2 receptor levels can be used to predict the prognosis of familial breast cancer patients. Prospectively identifying patients who are less likely to have non-familial breast cancer is vital for improving their overall survival.

The deletion of glucagon-like peptide-1 receptors expressing neurons in the dorsomedial hypothalamic nucleus disrupts the diurnal feeding pattern and induces hyperphagia and obesity.

BACKGROUND: Feeding rhythm disruption contributes to the development of obesity. The receptors of glucagon-like peptide-1 (GLP-1) are distributed in the wide regions of the brain. Among these regions, GLP-1 receptors (GLP-1R) are expressed in the dorsomedial hypothalamic nucleus (DMH) which are known to be associated with thermogenesis and circadian rhythm development. However, the physiological roles of GLP-1R expressing neurons in the DMH remain elusive. METHODS: To examine the physiological role of GLP-1R expressing neurons in the DMH, saporin-conjugated exenatide4 was injected into rat brain DMH to delete GLP-1R-positive neurons. Subsequently, locomotor activity, diurnal feeding pattern, amount of food intake and body weight were measured. RESULTS: This deletion of GLP-1R-positive neurons in the DMH induced hyperphagia, the disruption of diurnal feeding pattern, and obesity. The deletion of GLP-1R expressing neurons also reduced glutamic acid decarboxylase 67 and cholecystokinin A receptor mRNA levels in the DMH. Also, it reduced the c-fos expression after refeeding in the suprachiasmatic nucleus (SCN). Thirty percent of DMH neurons projecting to the SCN expressed GLP-1R. Functionally, refeeding after fasting induced c-fos expression in the SCN projecting neurons in the DMH. As for the projection to the DMH, neurons in the nucleus tractus solitarius (NTS) were found to be projecting to the DMH, with 33% of those neurons being GLP-1-positive. Refeeding induced c-fos expression in the DMH projecting neurons in the NTS. CONCLUSION: These findings suggest that GLP-1R expressing neurons in the DMH may mediate feeding termination. In addition, this meal signal may be transmitted to SCN neurons and change the neural activities.

Structure based analysis of KATP channel with a DEND syndrome mutation in murine skeletal muscle.

Developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, the most severe end of neonatal diabetes mellitus, is caused by mutation in the ATP-sensitive potassium (KATP) channel. In addition to diabetes, DEND patients present muscle weakness as one of the symptoms, and although the muscle weakness is considered to originate in the brain, the pathological effects of mutated KATP channels in skeletal muscle remain elusive. Here, we describe the local effects of the KATP channel on muscle by expressing the mutation present in the KATP channels of the DEND syndrome in the murine skeletal muscle cell line C2C12 in combination with computer simulation. The present study revealed that the DEND mutation can lead to a hyperpolarized state of the muscle cell membrane, and molecular dynamics simulations based on a recently reported high-resolution structure provide an explanation as to why the mutation reduces ATP sensitivity and reveal the changes in the local interactions between ATP molecules and the channel.

Identification of oxytocin receptor activating chemical components from traditional Japanese medicines.

Oxytocin (Oxt) is known to regulate social communication, stress and body weight. The activation of Oxt receptors (OTR) has clinical potential to abate stress disorders and metabolic syndrome. Kamikihito (KKT) is a traditional Japanese medicine used to treat psychological stress-related disorders. We investigated the effects of KKT, its ingredients and chemical components on Oxt neurons and OTR. C-Fos expression was examined after oral and peripheral administration of KKT in rats. Electrophysiological change of Oxt neurons and Oxt release upon application of KKT were measured in rat brain slice. The direct effect of KKT, its ingredients and its chemical components were examined by cytosolic Ca2+([Ca2+]i) measurement in Oxt neurons and OTR-expressing HEK293 cells. Both intraperitoneal and oral administration of KKT in rats induced c-Fos expression in neurons of the paraventricular nucleus (PVN) including Oxt neurons. Application of KKT induced activation of Oxt neurons and Oxt release. KKT increased [Ca2+]i in OTR-expressing HEK293 cells, and failed to activate with OTR antagonist. KKT-induced PVN Oxt neuron activation was also attenuated by OTR antagonist. Seven chemical components (rutin, ursolic acid, (Z )-butylidenephtalide, p-cymene, senkunolide, [6]-shogaol, [8]-shogaol) of three ingredients (Zizyphi Fructus, Angelicae Acutilobae Radix, Zingiberis Rhizoma) from KKT had potential to activate OTR. KKT can directly activate PVN Oxt neurons by interacting with OTR. The interaction of seven chemical components from KKT may contribute to activate OTR. Effect of KKT on Oxt neurons and OTR may contribute to the treatment of Oxt related disorders.

Structure of an antagonist-bound ghrelin receptor reveals possible ghrelin recognition mode.

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.

Early life high-fat diet exposure evokes normal weight obesity.

Obesity is becoming one of the most severe global health problems. However, risk of developing normal weight obesity, where an individual has a high percentage of body fat despite a normal body mass index, is gaining attention since such individuals also develop systemic inflammation and metabolic dysregulation. In this study, juvenile (3-week-old) and adult (8-week-old) rats were fed a high fat diet (HFD) for 9 weeks and compared them with normal chow diet (NCD) fed rats. The HFD fed adult group showed increase in energy intake, body weight (BW), total fat, visceral fat and subcutaneous fat compared with an age-matched NCD group. In addition, the percentage of muscle mass to BW in the adult HFD group was significantly lower compared with the NCD group. When HFD feeding was started from the juvenile stage, there were almost no differences in energy intake and BW between the HFD and NCD groups. However, the juvenile HFD group showed a 1.7-fold increase in total fat, visceral fat and subcutaneous fat compared with their age-matched NCD group. The percentage of muscle mass to BW was significantly lower in the juvenile HFD group compared with the NCD group. In addition, increased plasma insulin levels and decreased insulin sensitivity was observed only in juvenile HFD group, but not in adult HFD group. These results suggest that HFD feeding in growth period induces insulin resistance and normal weight obesity. Here we show a method for generating a normal weight obesity model, as well as raising the alarm for developing normal weight obesity when children are exposed to high-fat meals.

Goshajinkigan, a Traditional Japanese Medicine, Suppresses Voltage-Gated Sodium Channel Nav1.4 Currents in C2C12 Cells.

Goshajinkigan (GJG) is a traditional Japanese Kampo medicine used clinically to treat muscle pain in Japan. However, its underlying mechanism remains unclear. Since voltage-gated sodium channel (Nav) 1.4 is involved in skeletal muscle contraction, we investigated the possibility that GJG may affect Nav1.4 currents. By using an electrophysiological technique on skeletal muscle cell line C2C12, we found that GJG suppresses Nav1.4 currents in C2C12 cells. It is suggested that GJG may improve skeletal muscle stiffness or cramps by inhibiting abnormal Nav1.4 excitation. GJG may act as a Nav1.4 blocker and may be useful to treat muscle stiffness and clamps as well as easing the pain.

Oral oxytocin delivery with proton pump inhibitor pretreatment decreases food intake.

Oxytocin (Oxt) is considered as a potential agent to treat multiple neuropsychiatric disorders, obesity and metabolic syndrome. Although the mechanisms underlying these effects remain unclear, nasal administration is considered to be a potential way to deliver Oxt into blood vessels. The development of an easier, more stable and efficient way is expected. A recent study demonstrated that orally administered Oxt can be transmitted into blood if it is prevented from degradation in stomach and reaches the intestinal tract. In this study, we pretreated mice with a proton pump inhibitor (PPI), omeprazole (20 mg/kg), and administered capsulized Oxt (0.25 mg), so that the Oxt can be prevented from degradation by pepsin due to the low pH in stomach and reach the intestinal tract. Functionally, these mice showed a similar decrease in food intake to those who underwent intraperitoneal administration. We also confirmed that this method dramatically increased plasma Oxt levels and the expression of neural activation marker c-Fos protein in the paraventricular and suprachiasmatic nucleus. Our study showed that by pretreating mice with PPI, Oxt in a gelatin-coated capsule can prevent Oxt from degradation by pepsin in stomach, and reach the bloodstream in an effective concentration. These results indicate that our method is a promising oral delivery of Oxt and should be investigated further for other peptide agents based on peripheral injection or nasal administration.

TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice.

Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or various endogenous diseases related to bacterial translocation from the gut. Systemic inflammatory responses induced by endotoxemia induce severe involuntary loss of skeletal muscle, termed muscle wasting, which adversely affects the survival and functional outcomes of these patients. Currently, no drugs are available for the treatment of endotoxemia-induced skeletal muscle wasting. Here, we tested the effects of TAK-242, a Toll-like receptor 4 (TLR4)-specific signalling inhibitor, on myotube atrophy in vitro and muscle wasting in vivo induced by endotoxin. LPS treatment of murine C2C12 myotubes induced an inflammatory response (increased nuclear factor-κB activity and interleukin-6 and tumour necrosis factor-α expression) and activated the ubiquitin-proteasome and autophagy proteolytic pathways (increased atrogin-1/MAFbx, MuRF1, and LC-II expression), resulting in myotube atrophy. In mice, LPS injection increased the same inflammatory and proteolytic pathways in skeletal muscle and induced atrophy, resulting in reduced grip strength. Notably, pretreatment of cells or mice with TAK-242 reduced or reversed all the detrimental effects of LPS in vitro and in vivo. Collectively, our results indicate that pharmacological inhibition of TLR4 signalling may be a novel therapeutic intervention for endotoxemia-induced muscle wasting.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB-DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB-DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Cluster of differentiation 8 and programmed cell death ligand 1 expression in triple-negative breast cancer combined with autosomal dominant polycystic kidney disease and tuberous sclerosis complex: a case report.

BACKGROUND: Autosomal dominant polycystic kidney disease is defined as an inherited disorder characterized by renal cyst formation due to mutations in the PKD1 or PKD2 gene, whereas tuberous sclerosis complex is an autosomal dominant neurocutaneous syndrome caused by mutation or deletion of the TSC2 gene. A TSC2/PKD1 contiguous gene syndrome, which is caused by a chromosomal mutation that disrupts both the TSC2 and PKD1 genes, has been identified in patients with tuberous sclerosis complex and severe early-onset autosomal dominant polycystic kidney disease. The tumor tissue of patients with breast cancer with contiguous gene syndrome has a high mutation burden and produces several neoantigens. A diffuse positive immunohistochemistry staining for cluster of differentiation 8+ in the T cells of breast cancer tissue is consistent with neoantigen production due to high mutation burden. CASE PRESENTATION: A 61-year-old Japanese woman who had been undergoing dialysis for 23 years because of end-stage renal failure secondary to autosomal dominant polycystic kidney disease was diagnosed as having triple-negative breast cancer and underwent mastectomy in 2015. She had a history of epilepsy and skin hamartoma. Her grandmother, mother, two aunts, four cousins, and one brother were also on dialysis for autosomal dominant polycystic kidney disease. Her brother had epilepsy and a brain nodule. Another brother had a syndrome of kidney failure, intellectual disability, and diabetes mellitus, which seemed to be caused by mutation in the CREBBP gene. Immunohistochemistry of our patient's breast tissue showed cluster of differentiation 8 and programmed cell death ligand 1 positivity. CONCLUSIONS: Programmed cell death ligand 1 checkpoint therapy may be effective for recurrence of triple-negative breast cancer in a patient with autosomal dominant polycystic kidney disease and tuberous sclerosis complex.

The hypothalamus to brainstem circuit suppresses late-onset body weight gain.

Body weight (BW) is regulated in age-dependent manner; it continues to increase during growth period, and reaches a plateau once reaching adulthood. However, its underlying mechanism remains unknown. Regarding such mechanisms in the brain, we here report that neural circuits from the hypothalamus (paraventricular nucleus: PVN) to the brainstem (dorsal vagal complex: DVC) suppress late-onset BW gain without affecting food intake. The genetic suppression of the PVN-DVC circuit induced BW increase only in aged rats, indicating that this circuit contributes to suppress the BW at a fixed level after reaching adulthood. PVN neurons in the hypothalamus were inactive in younger rats but active in aged rats. The density of neuropeptide Y (NPY) terminal/fiber is reduced in the aged rat PVN area. The differences in neuronal activity, including oxytocin neurons in the PVN, were affected by the application of NPY or its receptor inhibitor, indicating that NPY is a possible regulator of this pathway. Our data provide new insights into understanding age-dependent BW regulation.

Association between cognitive impairment patient with solid cancer and insulin resistance.

OBJECTIVES: In an aging population, an increase in the number of elderly cancer patients with cognitive impairment is expected. The possible association between cancer and cognitive impairment is important to elucidate, because it can have a serious impact on quality of life. Here, we focused on glucose metabolism as a factor that links cancer and cognitive impairment. RESULTS: Thirteen subjects with solid cancers and cognitive impairment were recruited. As a control group, 14 subjects with cognitive impairment alone and 8 subjects with cancer alone were recruited. A Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and that of ß-cell function (HOMA-B) were used. In comparison with patients with solid cancer alone, those with cognitive impairment alone and those with both cancer and cognitive impairment had increased HOMA-IR values. Insulin resistance was increased in patients with cognitive impairment alone and those with both cognitive impairment and solid cancer than in patients without cognitive impairment; however, ß-cell function was not affected. The present data indicated that elderly cancer patients with high HOMA-IR score may be at a relatively high risk for developing cognitive impairment. Furthermore, early treatment to reduce insulin sensitivity may prevent cognitive impairment.

FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics.

Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.

The undeveloped properties of GABA neurons in the ventral tegmental area promote energy intake for growth in juvenile rats.

Juvenile animals show higher energy intake (EI) per body weight (BW) to meet the energy requirements for growth. However, the underlying mechanisms that induce high EI/BW in juvenile animals remain unknown. The EI from a control diet (CD) and high fat diet (HFD), as well as BW changes were compared between juvenile (3 weeks old) and adult (8 weeks old) rats. BW gain and EI were increased in the HFD-fed adult rats compared to the CD-fed adult rats. However, in the juvenile rats, there were no differences in BW gain and EI between the CD-fed and HFD-fed groups. The locomotor activity was significantly increased in HFD group compared with the CD group in juvenile, but not in adult rats. Gamma-aminobutyric acid (GABA) neurons in the VTA were found to remain undeveloped with less GABAergic input into dopamine neurons in the juvenile rats. The deletion of the VTA GABA neurons in the adult rats significantly increased CD consumption, but showed almost no change in HFD consumption. These data suggest that undeveloped properties of VTA GABA neurons in juvenile rats can promote higher EI regardless of high or less palatable feeding, and contribute to growth promotion.

MLH1 germline mutation associated with Lynch syndrome in a family followed for more than 45 years.

BACKGROUND: Lynch syndrome, is an autosomal dominantly inherited disease that predisposes individuals to a high risk of colorectal cancers, and some mismatch-repair genes have been identified as causative genes. The purpose of this study was to investigate the genomic rearrangement of the gene in a family with Lynch syndrome followed for more than 45 years. CASE PRESENTATION: The family with Lynch syndrome is family N, who received colorectal cancer treatment for 45 years. The proband of family N had multiple colorectal and uterine cancers. Because the proband met the diagnostic Amsterdam criteria and was Microsatellite instability (MSI) - positive, we performed genetic testing several times. However, germline mutations in MLH1 and MSH2 genes were not found by long-distance PCR or RT-PCR/direct sequencing analysis within the 45-year follow-up. MLPA analysis showed that the genomes of the proband and proband's daughter contained a deletion from exon 4 through exon 19 in the MLH1 gene. Her son's son and her daughter's son were found to be carriers of the mutation. CONCLUSIONS: For carriers of mismatch-repair gene mutation among families with Lynch syndrome, the onset risk of associated cancers such as uterine cancer is particularly high, including colorectal cancer. The diagnosis of carriers among non-onset relatives is important for disease surveillance.

Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds.

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

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 PGE2 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.