A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice.

A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+ ) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One-year-old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin-like growth factor-1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9-20-week-old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein-coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.

The Orphan GPR50 Receptor Regulates the Aggressiveness of Breast Cancer Stem-like Cells via Targeting the NF-kB Signaling Pathway.

The expression of GPR50 in CSLC and several breast cancer cell lines was assessed by RT-PCR and online platform (UALCAN, GEPIA, and R2 gene analysis). The role of GPR50 in driving CSLC, sphere formation, cell proliferation, and migration was performed using shGPR50 gene knockdown, and the role of GPR50-regulated signaling pathways was examined by Western blotting and Luciferase Assay. Herein, we confirmed that the expression of G protein-coupled receptor 50 (GPR50) in cancer stem-like cells (CSLC) is higher than that in other cancer cells. We examined that the knockdown of GPR50 in CSLC led to decreased cancer properties, such as sphere formation, cell proliferation, migration, and stemness. GPR50 silencing downregulates NF-kB signaling, which is involved in sphere formation and aggressiveness of CSLC. In addition, we demonstrated that GPR50 also regulates ADAM-17 activity by activating NOTCH signaling pathways through the AKT/SP1 axis in CSLC. Overall, we demonstrated a novel GPR50-mediated regulation of the NF-κB-Notch signaling pathway, which can provide insights into CSLC progression and prognosis, and NF-κB-NOTCH-based CSLC treatment strategies.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

GPR50 Distribution in the Mouse Cortex and Hippocampus.

G protein-coupled receptor 50 (GPR50) belongs to the G protein-coupled receptor which is highly homologous with the sequence of melatonin receptor MT1 and MT2. GPR50 expression has previously been reported in many brain regions, like cortex, midbrain, pons, amygdala. But, the distribution of GPR50 in the hippocampus and cortex and the cell types expressing GPR50 is not yet clear. In this study, we examined the distribution of GPR50 in adult male mice by immunofluorescence. Our results showed that GPR50 was localized in the CA1-3 pyramidal cells and the granule cells of the dentate gyrus. GPR50 was also expressed in excitatory and inhibitory neurons. As inhibitory neurons also contain many types, we found that GPR50 was localized in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Besides, similar results were seen in the cortex. The widespread expression of GPR50 in the hippocampus and cortex suggests that GPR50 may be associated with synaptic plasticity and cognitive function.

G protein coupled receptor 50 promotes self-renewal and neuronal differentiation of embryonic neural progenitor cells through regulation of notch and wnt/ß-catenin signalings.

G protein-coupled receptor 50 (GPR50), a risk factor for major depressive disorder and bipolar affective disorder, is expressed in both the developmental and adult brain. However, the function of GPR50 in the brain remains unknown. We here show GPR50 is expressed by neural progenitor cells (NPCs) in the ventricular zone of embryonic brain. Knockdown of GPR50 with a small interference RNA (siRNA) decreased self-renewal and neuronal differentiation, but not glial differentiation of NPCs. Moreover, overexpression of either full-length GPR50 or the intracellular domain of GPR50, rather than the truncated GPR50 in which the intracellular domain is deleted in, increased neuronal differentiation, indicating that GPR50 promotes neuronal differentiation of NPCs in an intracellular domain-dependent manner. We further described that the transcriptional activity of the intracellular domain of notch on Hes1 gene was repressed by overexpression of GPR50. In addition, decreased levels of transcription factor 7-like 2 (TCF7L2) mRNA was observed in GPR50 siRNA-transfected NPCs, suggesting that knockdown of GPR50 impairs wnt/ß-catenin signaling. Moreover, the mRNA levels of neurogenin (Ngn) 1, Ngn2 and cyclin D1, the target genes of notch and wnt/ß-catenin signalings, in NPCs were reduced by knockdown of GPR50. Therefore, GPR50 promotes self-renewal and neuronal differentiation of NPCs possibly through regulation of notch and wnt/ß-catenin signalings.

High expression of GPR50 promotes the proliferation, migration and autophagy of hepatocellular carcinoma cells in vitro.

G protein-coupled receptors (GPCRs) play important roles in tumorigenesis and the development of hepatocellular carcinoma (HCC). GPR50 is an orphan GPCR. Previous studies have indicated that GPR50 could protect against breast cancer development and decrease tumor growth in a xenograft mouse model. However, its role in HCC remains indistinct. To detect the role and the regulation mechanism of GPR50 in HCC, GPR50 expression was analyzed in HCC patients (gene expression omnibus database (GEO) (GSE45436)) and detected in HCC cell line CBRH-7919, and the results showed that GPR50 was significantly up-regulated in HCC patients and CBRH-7919 cell line compared to the corresponding normal control. Gpr50 cDNA was transfected into HCC cell line CBRH-7919, and we found that Gpr50 promoted the proliferation, migration, and autophagy of CBRH-7919. The regulation mechanism of GPR50 in HCC was detected by isobaric tags for relative and absolute quantification (iTRAQ) analysis, and we found that GPR50 promoted HCC was closely related to CCT6A and PGK1. Taken together, GPR50 may promote HCC progression via CCT6A-induced proliferation and PGK1-induced migration and autophagy, and GPR50 could be an important target for HCC.

Orphan receptor GPR50 attenuates inflammation and insulin signaling in 3T3-L1 preadipocytes.

Type 2 diabetes (T2DM) is characterized by insulin secretion deficiencies and systemic insulin resistance (IR) in adipose tissue, skeletal muscle, and the liver. Although the mechanism of T2DM is not yet fully known, inflammation and insulin resistance play a central role in the pathogenesis of T2DM. G protein-coupled receptors (GPCRs) are involved in endocrine and metabolic processes as well as many other physiological processes. GPR50 (G protein-coupled receptor 50) is an orphan GPCR that shares the highest sequence homology with melatonin receptors. The aim of this study was to investigate the effect of GPR50 on inflammation and insulin resistance in 3T3-L1 preadipocytes. GPR50 expression was observed to be significantly increased in the adipose tissue of obese T2DM mice, while GPR50 deficiency increased inflammation in 3T3-L1 cells and induced the phosphorylation of AKT and insulin receptor substrate (IRS) 1. Furthermore, GPR50 knockout in the 3T3-L1 cell line suppressed PPAR-γ expression. These data suggest that GPR50 can attenuate inflammatory levels and regulate insulin signaling in adipocytes. Furthermore, the effects are mediated through the regulation of the IRS1/AKT signaling pathway and PPAR-γ expression.

GPR50 participates in and promotes yak oocyte maturation: A new potential oocyte regulatory molecule.

Numerous molecular regulatory mechanisms are involved in the formation of mammalian oocytes, but many of their key proteins and molecules remain unknown. GPR50, the lone G protein-coupled receptor located in the X chromosome, is one of the superfamily members of the G protein-coupled receptor. GPR50 has been recently proved to play an important role in various physiological activities. However, the role of GPR50 in reproduction is currently unclear. In our previous research, we proved that the GPR50 receptor is present in yak oocytes. In the present study, the expression level and subcellular localization of GPR50 in the in vitro maturation process of yak oocytes were investigated to explore further its role in the maturation of yak oocytes. In the germinal vesicle (GV) stage, GPR50 was expressed and positioned in the cell membrane. By comparison, in the metaphase II (MII) stage, GPR50 had the highest expression level and was highly diffused in the cytoplasm. On the basis of these observations, the knockdown and overexpression of GPR50 yak oocyte models were constructed. Results showed that the expression level of GPR50 knockdown significantly reduced the excretion rate and maturity level of the yak oocyte PB1 (P < 0.01). However, the expression level of GPR50 overexpression exerted no significant influence on the excretion rate and maturity level of the yak oocytes (P > 0.05). This study verified that GPR50 plays an important role in the in vitro maturation process of yak oocytes.

ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6 h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50, upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

The orphan GPR50 receptor interacting with TßRI induces G1/S-phase cell cycle arrest via Smad3-p27/p21 in BRL-3A cells.

The liver has the powerful capacity to regenerate after injury or resection. In one of our previous studies, GPR50 was observed to be significantly upregulated at 6 h, following a partial hepatectomy (PH) in rat liver regeneration (LR) via gene expression profile. However, little research has been done on the regulation and mechanism of GPR50 in the liver. Herein, we observed that the overexpression of GPR50 inhibited the proliferation of BRL-3A cells. To further explore the molecular mechanisms of GPR50 in the regulation of BRL-3A cell proliferation, interaction between GPR50 and transforming growth factor-beta I (TßRI) and iTRAQTM differential proteomic analysis were elucidated, which suggested that GPR50 may interact with TßRI to activate the TGF-ß signaling pathway and arrest BRL-3A cell cycle G1/S transition. Subsequently, the potential mechanism underlying the role of GPR50 in hepatocyte growth was also explored through the addition of a signaling pathway inhibitor. These data suggested that interaction between the orphan GPR50 receptor and TßRI induced the G1/S-phase cell cycle arrest of BRL-3A cells via the Smad3-p27/p21 pathway.

Measuring Protein-Protein Interactions of Melatonin Receptors by Bioluminescence Resonance Energy Transfer (BRET).

The melatonin receptor subfamily belongs to the G protein-coupled receptor superfamily and consists of three members in mammals, MT1, MT2, and GPR50. These receptors can interact with each other to form homo- and heterodimers that are part of larger molecular complexes composed of G proteins, ß-arrestins, and other membrane and cytosolic proteins. BRET (bioluminescence resonance energy transfer) is a versatile technique to follow protein-protein interactions on the nanometer scale, in real time, in living cells, which contributed largely to our understanding of the function of melatonin receptors. In this chapter, we describe our BRET protocols for melatonin receptors, which can also be applied to other GPCRs.

GPR50 Promotes Hepatocellular Carcinoma Progression via the Notch Signaling Pathway through Direct Interaction with ADAM17.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and it is thus critical to identify novel molecular biomarkers of HCC prognosis and elucidate the molecular mechanisms underlying HCC progression. Here, we show that G-protein-coupled receptor 50 (GPR50) in HCC is overexpressed and that GPR50 knockdown may downregulate cancer cell progression through attenuation of the Notch signaling pathway. GPR50 knockdown was found to reduce HCC progression by inactivating Notch signaling in a ligand-independent manner through a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), a proteolytic enzyme that cleaves the Notch receptor, which was corroborated by GPR50 overexpression in hepatocytes. GPR50 silencing also downregulated transcription and translation of ADAM17 through the AKT/specificity protein-1 (SP1) signaling axis. Notably, GPR50 was found to directly interact with ADAM17. Overall, we demonstrate a novel GPR50-mediated regulation of the ADAM17-Notch signaling pathway, which can provide insights into HCC progression and prognosis and development of Notch-based HCC treatment strategies.

Melatonin Receptor Signaling: Impact of Receptor Oligomerization on Receptor Function.

The melatonin receptor subfamily is composed of three members, MT1 and MT2, which are binding to melatonin, and GPR50, which shows high sequence homology to MT1 and MT2 but does not bind to melatonin or any other known ligand. An interesting feature of these receptors is their capacity to form homo- and heteromers between each other and also with other GPCRs. The following heteromers have been described: MT1/MT2, MT1/GPR50, and heteromers composed of MT2 and the serotonin 5-HT2c receptor or the orphan GPR61, GPR62, and GPR135 receptors. These heteromers represent novel pharmacological entities as they exhibit functional properties that are different from those of the corresponding homomers. Formation of several of these heteromers has been confirmed in tissues. MT2/5-HT2c heteromers are targeted by the clinically relevant antidepressant agomelatine, and MT1/MT2 heteromers regulate nocturnal retinal light sensitivity. Here, we resume our current knowledge on melatonin receptor heteromerization and discuss how it contributes to the diversification of the function of melatonin receptors.

Alternation between short- and long photoperiod reveals hypothalamic gene regulation linked to seasonal body weight changes in Djungarian hamsters (Phodopus sungorus).

Djungarian hamsters are able to reduce their body weight by more than 30% in anticipation of the winter season. This particular adaptation to extreme environmental conditions is primarily driven by a natural reduction in day length and conserved under laboratory conditions. We used this animal model to investigate hypothalamic gene expression linked to body weight regulation behind this physiological phenomenon. After an initial collective short photoperiod (SP) adaptation for 14 weeks from a preceding long photoperiod (LP), hamsters were re-exposed to LP for either 6 or 14 weeks, followed by a second re-exposure to SP for 8 weeks. Our data showed that re-exposure to LP led to an increase in body weight. In the hypothalamus Dio2, Vimentin, Crbp1 and Grp50 expression increased, whereas expression of Dio3, Mct8 and Srif decreased. The changes in body weight and gene expression were reversible in most hamsters after a further re-exposure to SP following 6 or 14 weeks in LP. Interestingly, after 14 weeks in LP, body weight loss was pronounced in six hamsters re-exposed to SP, but five hamsters did not respond. In nonresponding hamsters, a different gene expression pattern was manifested, with the exception of Dio2, which was reduced not only in SP re-exposed hamsters, but also in hamsters maintained in LP. Taken together, these data suggest that body weight regulation appears to be tightly linked to a co-ordinated regulation of several genes in the hypothalamus, including those involved in thyroid hormone metabolism.

The emerging role of GPR50 receptor in brain.

GPR50 receptor one of the member of G protein-coupled receptors (GPCRs) is extensively expressed in the pituitary, hypothalamus,cortex, midbrain, pons, amygdala, and in several brainstem nuclei. The exact function of this receptor in brain is remains unclear. This review presents current knowledge regarding the function of GPR50 receptor in brain, with a focus on role of this receptor in the hypothalamus-pituitary-adrenal (HPA) axis and the glucocorticoid receptor (GR) signaling, leptin signaling, adaptive thermogenesis, torpor, neurite outgrowth, and self-renewal and neuronal differentiation of neural progenitor cells NPCs. Although the results are encouraging, further research is needed to clarify GPR50 role in neurobiology of mood disorders, adaptive thermogenesis, torpor, and in the pathophysiology of neurological disorders.

Resveratrol: brain effects on SIRT1, GPR50 and photoperiodic signaling.

Silent information regulator-1 (SIRT1) deacetylase, a sensor of intermittent energy restriction, is inextricably intertwined with circadian regulation of central and peripheral clock genes. The purpose of this study was to identify SIRT1-specific target genes that are expressed in a circadian rhythm pattern and driven, in part, by specific components of foodstuffs. Using human cells and rats fed with a resveratrol diet we show that SIRT1 binds to, and transcriptionally regulates, a gene locus encoding the G protein-coupled receptor (GPR), GPR50 in the brain. GPR50 is the mammalian orthologue of the melatonin1c membrane-bound receptor which has been identified as a genetic risk factor for bipolar disorder and major depression in women. In general, our findings support and expand the notion that circadian clock signaling components and dietary interventions are adaptively linked, and suggest that the brain may be particularly sensitive to metabolic events in response to light-dark cycles.

Involvement of GPR50 polymorphisms in depression: independent replication in a prospective elderly cohort.

INTRODUCTION: Despite the explosion in genetic association studies over the last decade, clearly identified genetic risk factors for depression remain scarce and replication studies are becoming increasingly important. G-protein-coupled receptor 50 (GPR50) has been implicated in psychiatric disorders in a small number of studies, although not consistently. METHODS: Data were obtained from 1010 elderly men and women from the prospective population-based ESPRIT study. Logistic regression and survival models were used to determine whether three common GPR50 polymorphisms were associated with depression prevalence or the incidence of depression over 12-years. The analyses were adjusted for a range of covariates such as comorbidity and cholesterol levels, to determine independent associations. RESULTS: All three variants showed some evidence of an association with late-life depression in women, although these were not consistent across outcomes, the overall effect sizes were relatively small, and most would not remain significant after correction for multiple testing. Women heterozygous for rs13440581, had a 1.6-fold increased risk of baseline depression, while the odds of depression comorbid with anxiety were increased fourfold for women homozygous for the minor allele of rs2072621. When depressed women at baseline were excluded from the analysis, however, neither variant was associated with the 12-year incidence of depression. In contrast, rs561077 was associated with a 1.8-fold increased risk of incident depression specifically. No significant associations were observed in men. DISCUSSION: Our results thus provide only weak support for the involvement of GPR50 variants in late-life depression, which appear specific to certain subgroups of depressed individuals (i.e., women and those with more severe forms of depression).