Progestin And adipoQ Receptor Research Articles

TrhA, a bacterial progestin and adiponectin receptor homolog, couples membrane energetics homeostasis and unsaturated fatty acid metabolism.

Members of the widely conserved progestin and adipoQ receptor (PAQR) family function to maintain membrane homeostasis: membrane fluidity and fatty acid composition in eukaryotes and membrane energetics and fatty acid composition in bacteria. All PAQRs consist of a core seven transmembrane domain structure and five conserved amino acids (three histidines, one serine, and one aspartic acid) predicted to form a hydrolase-like catalytic site. PAQR homologs in Bacteria (called TrhA, for transmembrane homeostasis protein A) maintain homeostasis of membrane charge gradients, like the membrane potential and proton gradient that comprise the proton motive force, but their molecular mechanisms are not yet understood. Here, we show that TrhA in Escherichia coli has a periplasmic C-terminus, which places the five conserved residues shared by all PAQRs at the cytoplasmic interface of the membrane. Here, we characterize several conserved residues predicted to form an active site by site-directed mutagenesis. We also identify a specific role for TrhA in modulating unsaturated fatty acid biosynthesis with conserved residues required to either promote or reduce the abundance of unsaturated fatty acids. We also identify distinct roles for the conserved residues in supporting TrhA's role in maintaining membrane energetics homeostasis that suggest that both functions are intertwined and probably partly dependent on one another. An analysis of domain architecture of TrhA-like domains in Bacteria further supports a function of TrhA linking membrane energetics homeostasis with biosynthesis of unsaturated fatty acid in the membrane. IMPORTANCE Progestin and adipoQ receptor (PAQR) family proteins are evolutionary conserved regulators of membrane homeostasis and have been best characterized in eukaryotes. Bacterial PAQR homologs, named TrhA (transmembrane homeostasis protein A), regulate membrane energetics homeostasis through an unknown mechanism. Here, we present evidence linking TrhA to both membrane energetics homeostasis and unsaturated fatty acid biosynthesis. Analysis of domain architecture together with experimental evidence suggests a model where TrhA activity on unsaturated fatty acid biosynthesis is regulated by changes in membrane energetics to dynamically adjust membrane homeostasis.

Integrative pan-cancer analysis reveals the importance of PAQR family in lung cancer.

The progestin and adipoQ receptors (PAQRs) family contains 11 genes involved in the regulation of metabolism and cancer development. However, a comprehensive understanding of the role of PAQRs in cancer remains largely scarce, and the associations between their expression levels and immune signatures also need to be researched. Here, we applied pan-cancer analysis to explore the associations between PAQRs expression and survival, tumor microenvironment (TME), and drug sensitivity from the UCSC Xena and CellMiner databases. Besides, we further studied the expression, survival and somatic mutations of PAQRs in lung cancer (LC) from TCGA database. The results showed that PAQRs had significant heterogeneity with some upregulation and some downregulation in most tumors. Specifically, compared with PAQR3/5/6/9 and MMD2, ADIPOR1/2, PAQR4/7/8 and MMD had higher levels of average expression in all tumor types. PAQRs expression was greatly correlated with survival, immune subtypes, TME, and drug sensitivity. Furthermore, this research concentrated on analyzing the relationship of PAQRs expression with LC prognosis, and proved that ADIPOR2, PAQR4/9 and MMD were independent prognostic factors for LC patients. Finally, based on somatic mutation data, the genetic mutations in LC patients were majorly missense mutations, and TP53 and TTN had the top two highest mutation frequencies. Collectively, PAQRs may serve as robust biomarkers to predict the prognosis and guide immunotherapy of tumors, especially LC, which enables novel ways for improving cancer treatment.

MPRα and PR co-operate in progesterone inhibition of endothelial cell focal adhesion.

Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.

Structure and function analyses of the Mmd2 gene in pacific white shrimp Litopenaeus vannamei.

Monocyte to macrophage differentiation factor 2 gene (Mmd2) encodes a member of the progestin and adipoQ receptor (PAQR) family, and plays a key role in growth and development. Our previous studies had found Mmd2 (Monocyte to macrophage differentiation factor 2) is a new candidate gene for growth traits in Pacific white shrimp (Litopenaeus vannamei). For the purpose of understanding the underlying mechanism of LvMmd2 affecting the growth of shrimp, we analyzed the gene structure, phylogeny, expression profiles and RNA interference of this gene in L. vannamei. We found the LvMmd2 gene sequence was highly conserved in transmembrane regions, it was widely expressed in different tissues, with the highest expression level in the eye stalk. Knockdown LvMmd2 could significantly promote body length and body weight gain, suggesting it is a growth suppressor. Through transcriptome analysis we identified 422 differentially expressed genes (DEGs) between the dsMmd2 group and control group, among which 337 genes were upregulated in the dsMmd2 group, including numerous muscle-related genes and protein synthesis genes. Further bioinformatics analysis showed that growth, metabolism, and immune-related signal pathway had changed significantly. The above results greatly increase our understanding on the conservative structure and function of LvMmd2 gene, and provide potential application prospects in genetics and breeding.

Membrane Progesterone Receptors (mPRs, PAQRs): Review of Structural and Signaling Characteristics.

The role of membrane progesterone receptors (mPRs), which belong to the progestin and adipoQ receptor (PAQR) family, in mediating rapid, nongenomic (non-classical) progestogen actions has been extensively studied since their identification 20 years ago. Although the mPRs have been implicated in progestogen regulation of numerous reproductive and non-reproductive functions in vertebrates, several critical aspects of their structure and signaling functions have been unresolved until recently and remain the subject of considerable debate. This paper briefly reviews recent developments in our understanding of the structure and functional characteristics of mPRs. The proposed membrane topology of mPRα, the structure of its ligand-binding site, and the binding affinities of steroids were predicted from homology modeling based on the structures of other PAQRs, adiponectin receptors, and confirmed by mutational analysis and ligand-binding assays. Extensive data demonstrating that mPR-dependent progestogen regulation of intracellular signaling through mPRs is mediated by activation of G proteins are reviewed. Close association of mPRα with progesterone membrane receptor component 1 (PGRMC1), its role as an adaptor protein to mediate cell-surface expression of mPRα and mPRα-dependent progestogen signaling has been demonstrated in several vertebrate models. In addition, evidence is presented that mPRs can regulate the activity of other hormone receptors.

Functional Analysis of Mmd2 and Related PAQR Genes During Sex Determination in Mice

Introduction: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. Mmd2 is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. Methods: We used CRISPR to generate mouse strains deficient in Mmd2 and its 2 closely related PAQR family members, Mmd and Paqr8, which are also expressed during testis development. Following characterization of Mmd2 expression in the developing testis, we studied sex determination in embryos from single knockout as well as Mmd2;Mmd and Mmd2;Paqr8 double knockout lines using quantitative RT-PCR and immunofluorescence. Results: Analysis of knockout mice deficient in Sox9 and Nr5a1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in Mmd2-null embryos. To determine if other genes might have compensated for the loss of Mmd2, we analyzed Paqr8 and Mmd-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. Discussion: These results may reflect functional redundancy among PAQR factors, or their dispensability in gonadal development. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

Bacterial Homologs of Progestin and AdipoQ Receptors (PAQRs) Affect Membrane Energetics Homeostasis but Not Fluidity.

ABSTRACTMembrane potential homeostasis is essential for cell survival. Defects in membrane potential lead to pleiotropic phenotypes, consistent with the central role of membrane energetics in cell physiology. Homologs of the progestin and AdipoQ receptors (PAQRs) are conserved in multiple phyla of Bacteria and Eukarya. In eukaryotes, PAQRs are proposed to modulate membrane fluidity and fatty acid (FA) metabolism. The role of bacterial homologs has not been elucidated. Here, we use Escherichia coli and Bacillus subtilis to show that bacterial PAQR homologs, which we name “TrhA,” have a role in membrane energetics homeostasis. Using transcriptional fusions, we show that E. coli TrhA (encoded by yqfA) is part of the unsaturated fatty acid biosynthesis regulon. Fatty acid analyses and physiological assays show that a lack of TrhA in both E. coli and B. subtilis (encoded by yplQ) provokes subtle but consistent changes in membrane fatty acid profiles that do not translate to control of membrane fluidity. Instead, membrane proteomics in E. coli suggested a disrupted energy metabolism and dysregulated membrane energetics in the mutant, though it grew similarly to its parent. These changes translated into a disturbed membrane potential in the mutant relative to its parent under various growth conditions. Similar dysregulation of membrane energetics was observed in a different E. coli strain and in the distantly related B. subtilis. Together, our findings are consistent with a role for TrhA in membrane energetics homeostasis, through a mechanism that remains to be elucidated.IMPORTANCE Eukaryotic homologs of the progestin and AdipoQ receptor family (PAQR) have been shown to regulate membrane fluidity by affecting, through unknown mechanisms, unsaturated fatty acid (FA) metabolism. The bacterial homologs studied here mediate small and consistent changes in unsaturated FA metabolism that do not seem to impact membrane fluidity but, rather, alter membrane energetics homeostasis. Together, the findings here suggest that bacterial and eukaryotic PAQRs share functions in maintaining membrane homeostasis (fluidity in eukaryotes and energetics for bacteria with TrhA homologs).

PAQR6 Upregulation Is Associated with AR Signaling and Unfavorite Prognosis in Prostate Cancers.

Progesterone-induced rapid non-genomic signaling events have been confirmed through several membrane progesterone receptors (mPR). Some mPRs were reported to correlate with cancer progression and patient prognosis. In this study, we conducted a comprehensive analysis of all progesterone receptor (PGR)-related genes in prostate cancer tissues and examined the correlations of their expression levels with disease progression and patient survival outcomes. We utilized multiple RNA-seq and cDNA microarray datasets to analyze gene expression profiles and performed logistics aggression and Kaplan-Meier survival analysis after stratifying patients based on tumor stages and Gleason scores. We also used NCBI GEO datasets to examine gene expression patterns in individual cell types of the prostate gland and to determine the androgen-induced alteration of gene expression. Spearman coefficient analysis was conducted to access the correlation of target gene expression with treatment responses and disease progression status. The classic PGR was mainly expressed in stromal cells and progestin and adipoQ receptor (PAQR) genes were the predominant genes in prostate epithelial cells. Progesterone receptor membrane component-1 (PGRMC1) was significantly higher than PGRMC2 in all prostate cell types. In prostate cancer tissues, PAQR6 expression was significantly upregulated, while all other genes were largely downregulated compared to normal prostate tissues. Although both PAQR6 upregulation and PAQR5 downregulation were significantly correlated with tumor pathological stages, only PAQR6 upregulation was associated with Gleason score, free-prostate-specific antigen (fPSA)/total-PSA (tPSA) ratio, and patient overall survival outcomes. In addition, PAQR6 upregulation and PGR/PGRMC1 downregulation were significantly associated with a quick relapse. Conversely, in neuroendocrinal prostate cancer (NEPC) tissues, PAQR6 expression was significantly lower, but PAQR7/8 expression was higher than castration-resistant prostate cancer (CRPC) tissues. PAQR8 expression was positively correlated with androgen receptor (AR) score and AR-V7 expression levels but inversely correlated with NEPC score in metastatic CRPC tumors. This study provides detailed expression profiles of membrane progesterone receptor genes in primary cancer, CRPC, and NEPC tissues. PAQR6 upregulation in primary cancer tissues is a novel prognostic biomarker for disease progression, overall, and progression-free survival in prostate cancers. PAQR8 expression in CRPC tissues is a biomarker for AR activation.

PAQR7: An intermediary mediating nongenomic progesterone action in female reproductive tissue

Progestin and adipoQ receptor 7 (PAQR7) as an indispensable member of membrane progestin receptors in the Progestin and adipoQ receptor (PAQR) family that mediates nongenomic progesterone actions, initiated rapidly at the cell surface. Previous research demonstrated the distribution of PAQR7, which was mainly expressed in reproductive tissues, including ovary and testis. In the male reproductive system, PAQR7 is involved in progestin-induced sperm hypermotility. However, reports studying PAQR7 in female reproductive tissue mainly concentrate on oocyte maturation in fish, its expression in the ovary and gestational tissue, and regulation of uterine functions in mammals. Despite recent advances, many aspects of progestin signaling through PAQR7 are still unclear, especially in female reproductive tissue. Therefore, we reveal the structure and characteristics of PAQR7 and conclude the putative progestin-induced action mediated by PAQR7 in female reproductive tissue, such as the development of ovarian follicles, apoptosis of granulosa cells, oocyte maturation, and development of certain diseases, among others, to review the function of PAQR7 in the female reproductive system in detail.

Anti-apoptotic Actions of Allopregnanolone and Ganaxolone Mediated Through Membrane Progesterone Receptors (PAQRs) in Neuronal Cells.

The neurosteroids progesterone and allopregnanolone regulate numerous neuroprotective functions in neural tissues including inhibition of epileptic seizures and cell death. Many of progesterone's actions are mediated through the nuclear progesterone receptor (PR), while allopregnanolone is widely considered to be devoid of hormonal activity and instead acts through modulation of GABA-A receptor activity. However, allopregnanolone can also exert hormonal actions in neuronal cells through binding and activating membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) family. The distribution and functions of the five mPR subtypes (α, β, γ, δ, ε) in neural tissues are briefly reviewed. mPRδ has the highest binding affinity for allopregnanolone and is highly expressed throughout the human brain. Low concentrations (20 nM) of allopregnanolone act through mPRδ to stimulate G protein (Gs)-dependent signaling pathways resulting in reduced cell death and apoptosis in mPRδ-transfected cells. The 3-methylated synthetic analog of allopregnanolone, ganaxolone, is currently undergoing clinical trials as a promising GABA-A receptor-selective antiepileptic drug (AED). New data show that low concentrations (20 nM) of ganaxolone also activate mPRδ signaling and exert anti-apoptotic actions through this receptor. Preliminary evidence suggests that ganaxolone can also exert neuroprotective effects by activating inhibitory G protein (Gi)-dependent signaling through mPRα and/or mPRβ in neuronal cells. The results indicate that mPRs are likely intermediaries in multiple actions of natural and synthetic neurosteroids in the brain. Potential off-target effects of ganaxolone through activation of mPRs in patients receiving long-term treatment for epilepsy and other disorders should be considered and warrant further investigation.

SAT-739 Membrane Progesterone Receptors (mPRs/PAQRs) Promote Proliferation, Migration and Neurotrophin Expression in Schwann Cell-like Adipose Stem Cells, Potentially Improving Peripheral Nerve Regeneration

Peripheral nerve injury is a problem affecting millions of people worldwide, causing significant disability and a reduction in the quality of life. The neurons of the peripheral nervous system are characterized by a significant regeneration capability. However, the functional recovery is unsatisfactory in most cases, making the identification of new strategies to improve the regenerative outcome a major medical need. Schwann cells (SCs), the main neuroglial cells of the peripheral nervous system, trans-differentiate after nerve injury towards a phenotype, known as the repair phenotype, that promotes nerve regeneration. Nonetheless, adult human SCs have poor expansion capability in vitro, undermining their potential clinical development. Several studies have shown that mesenchymal stem cells, especially when differentiated into a Schwann cell-like (SCL) phenotype, may represent an alternative to primary SCs, promoting pro-regenerative effects both in vitro and in vivo. Recently, membrane progesterone receptors (mPRs), members of the progestin and adipoQ receptor (PAQR) family, have been shown to be present and active in Schwann cells. Progesterone activates mPRs in SCs to promote cell migration and proliferation, change cell morphology, and modulate the expression of key SC differentiation factors. These findings suggest a possible role for mPRs in nerve repair. The goal of the present project is to study the role of mPRs in the promotion of pro-regenerative effects in mesenchymal adipose stem cells (ASC), both undifferentiated (uASC) and after differentiation toward the SCL phenotype (SCL-ASC). We first characterized uASC and SCL-ASC, confirming that the latter showed increased expression of SC markers like S100B, Sox10, GFAP, P0 and Krox20, alongside a more pronounced spindle-like shape leading to an increased aspect ratio. The differentiation protocol also increased mPR gene expression in SCL-ASC compared to uASC, especially mPRα (PAQR7) and mPRβ (PAQR8). Treatment with the specific mPR agonist Org OD 02-0 (02-0) caused cell elongation in SCL-ASC, mimicking what happens to SCs in vivo when they trans-differentiate towards the repair phenotype. 02-0 treatment also led to increased cell migration and cell proliferation in both uASC and SCL-ASC, consistent with a pro-regenerative role of mPRs. The effect was more rapid and evident in SCL-ASC. Lastly, we analyzed the effect of mPR activation on neutrophin production and release in uASC and SCL-ASC. A significant increase was observed in the expression and release of the brain derived neurotrophic factor (BDNF), whose role in promoting neurogenesis is well established. Our results support the hypothesis that mPRs have an important role in the modulation of uASC and SCL-ASC physiology and their activation may promote nerve regeneration.

PAQR4 promotes chemoresistance in non-small cell lung cancer through inhibiting Nrf2 protein degradation.

Purpose: Lung cancer is the leading cause of cancer related deaths worldwide. We have previously identified many differentially expressed genes (DEGs) from large scale pan-cancer dataset using the Cross-Value Association Analysis (CVAA) method. Here we focus on Progestin and AdipoQ Receptor 4 (PAQR4), a member of the progestin and adipoQ receptor (PAQR) family localized in the Golgi apparatus, to determine their clinical role and mechanism in the development of non-small cell lung cancer (NSCLC).Methods: The protein expression profile of PAQR4 was examined by IHC using tissue microarrays, and the effects of PAQR4 on cell proliferation, colony formation and xenograft tumor formation were tested in NSCLC cells. Real-time RT-PCR, co-immunoprecipitation (co-IP) and GST-pulldown assays were used to explore the mechanism of action of PAQR4.Results: We provided evidence showing that PAQR4 is increased in NSCLC cancer cell lines (A549, H1299, H1650, H1975, H358, GLC-82 and SPC-A1), and identified many mutations in PAQR4 in non-small cell lung cancer (NSCLC) tissues. We demonstrated that PAQR4 high expression correlates with a worse clinical outcome, and that its knockdown suppresses cell proliferation by inducing apoptosis. Importantly, overexpressed PAQR4 physically interacts with Nrf2 in NSCLC cells, blocking the interaction between Nrf2 and Keap1.Conclusion: Our results suggest that PAQR4 depletion enhances the sensitivity of cancerous cell to chemotherapy both in vitro and xenograft tumor formation in vivo, by promoting Nrf2 protein degradation through a Keap1-mediated ubiquitination process.

Characterization of the Golgi scaffold protein PAQR3, and its role in tumor suppression and metabolic pathway compartmentalization.

The Golgi apparatus is critical in the compartmentalization of signaling cascades originating from the cytoplasmic membrane and various organelles. Scaffold proteins, such as progestin and adipoQ receptor (PAQR)3, specifically regulate this process, and have recently been identified in the Golgi apparatus. PAQR3 belongs to the PAQR family, and was recently described as a tumor suppressor. Accumulating evidence demonstrates PAQR3 is downregulated in different cancers to suppress its inhibitory effects on malignant potential. PAQR3 functions biologically through the pathological regulation of altered signaling pathways. Significant cell proliferation networks, including Ras proto-oncogene (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), insulin, and vascular endothelial growth factor, are closely controlled by PAQR3 for physiologically relevant effects. Meanwhile, genetic/epigenetic susceptibility and environmental factors, may have functions in the downregulation of PAQR3 in human cancers. This study aimed to assess the subcellular localization of PAQR3 and determine its topological features and functional domains, summarizing its effects on cell signaling compartmentalization. The pathophysiological functions of PAQR3 in cancer pathogenesis, metabolic diseases, and developmental ailments were also highlighted.

In Vitro Effects of Coal and Progesterone on Glioblastoma Proliferation and Progestogen Receptor Expression

Glioma incidence in males is 1.5 times that of females, thus it is suspected that female sex hormones play a protective role against gliomas. Compounds found in water contaminated by extractive industry waste can disrupt hormone mediated signaling. Studies have been done on endocrine disrupting effects on estrogen signaling, but little has been done on their effects on progesterone signaling. Additionally, the focus has been on classical receptors, with almost no investigation of G-protein coupled receptors. Here we investigate the effects of exposure to coal dust on cell proliferation and expression of progestin and adipoQ receptors (PAQRs) in two human glioma cell lines, A172 and U87. Treatments with coal dust and treatments with progesterone were performed. End point RT-PCR demonstrated that A172 cells express PAQR3, PAQR5, and PAQR9. This was the first demonstration of PAQR5 expression in A172 cells. U87 cells express PAQR3, PAQR5, PAQR7, PAQR8, and PAQR9. Real time RT-PCR was performed to determine relative receptor expression per treatment type. U87 cells treated with coal dust showed up-regulation of PAQR8 and PAQR9. A172 cells treated with either progesterone or coal dust showed up-regulation of PAQR9. Proliferation test on A172 showed 20% increase in proliferation in cells treated with coal dust for 24 hours and a 6% increase in cells treated for 48 hours. The U87 cell line showed a 7% increase in proliferation after 24 hours of coal dust treatment, with no statistically significant change for 48-hour treatments. Neither cell line expressed statistically significant changes after progesterone treatment. Supported by NSF EPSCoR Research Infrastructure Improvement Program award 1458952, the NASA West Virginia Space Grant Consortium, and NIH Grants 5P20RR016477 and P20GM103434 to the West Virginia IDeA Network for BioMedical Research Funding.

Membrane progesterone receptors β and γ have potential as prognostic biomarkers of endometrial cancer

Endometrial cancer (EC) is one of the most common malignancies in women worldwide. EC is linked to chronic exposure to estrogens that is unopposed by protective effects of progesterone. Progesterone modulates gene expression via classical nuclear receptors, and has rapid effects via the less characterized membrane-bound progesterone receptors (mPRs) of the progestin and adipoQ receptor (PAQR) family. The presence of mPRs in EC has not been investigated to date. The aims of this study were to examine PAQR7, PAQR8 and PAQR5, which encode for mPRα, mPRβ and mPRγ, respectively, for their expression and localization in EC tissue and adjacent control endometrium. Our results reveal decreased expression of PAQR7 and PAQR8, and unaltered expression of PAQR5 in EC versus control tissue. Expression of PAQR5 was decreased in EC with higher FIGO stage versus stage IA. Immunohistochemistry revealed lower levels of mPRα and mPRβ, but higher levels of mPRγ, in EC versus control tissue. There was greater decrease in mPRβ levels in tumors with lymphovascular invasion. The analysis of the expression data associates higher PAQR5 mRNA and mPRβ protein levels with favorable patient prognosis. Immunohistochemistry showed diverse localizations of mPRs in control and cancer endometrium. In control endometrium, mPRα and mPRβ were localized mostly at the cell membranes, while mPRγ was localized in the cytoplasm and/or nucleus. In cancer endometrium, mPRα and mPRβ were detected at the cell membrane or in the cytoplasm, or both, while mPRγ was only localized in the cytoplasm. Taken together, these results imply that mPRs are involved in EC pathogenesis through effects on the development or progression of cancer. The potential role of mPRβ and mPRγ as prognostic biomarkers needs to be further assessed on a larger number of samples.

Identification and characterization of a progestin and adipoQ receptor (PAQR) structurally related to Paqr7 in the ovary of Cynoglossus semilaevis and its potential role in regulating oocyte maturation

Membrane progestin receptors (mPRs) play an important role in the regulation of oocyte meiotic maturation in fish. However, details of the molecular endocrine mechanism regulating oocyte maturation in multiple spawning fish with asynchronous ovarian development remain unclear. The cDNA encoding a novel progestin and adipoQ receptor with structural similarity to mPRα (Paqr7), herein called Paqr7b, was cloned and sequenced from the ovary of half-smooth tongue sole Cynoglossus semilaevis. Phylogenetic analysis showed that Paqr7b represents an evolutionary intermediate between mPRα and mPRβ and shares high homology with other similar Paqr proteins in other teleost species. However, the tongue sole Paqr7b protein showed much greater homology to teleost mPRαs (average 52%) than mPRβs (average 40%), suggesting it may have arisen from gene duplication of mPRα. paqr7b and paqr7 mRNA exhibited similar patterns of tissue expression. The mRNA and protein of Paqr7b were ubiquitously detected in all tissues analyzed, including the ovary. Moreover, in situ hybridization results revealed that paqr7b was expressed in stage V oocytes, as well as in scattered cells in the pituitary. The expression of paqr7b mRNA in brain and ovary significantly increased from ovarian development stage II to stage V (P<0.05), and was maximal at stage V, and then sharply decreased at stage VI. The transcript level of paqr7b mRNA in the pituitary also peaked at stage V (P<0.05). Treatment of tongue sole ovarian follicles with gonadotropin consistently increased the expression level of Paqr7b protein and mRNA in both a dose- and stage-dependent manner. Microinjection of tongue sole oocytes with a morpholino antisense oligonucleotide to Paqr7b blocked the progestin induction of oocyte maturation. Our findings demonstrate an important role of Paqr7b in the regulation of oocyte maturation in tongue sole and suggest the receptor may also influence other aspects of reproduction, such as pituitary function.

Molecular characterization of an adiponectin receptor homolog in the white leg shrimp, Litopenaeus vannamei.

Adiponectin (AdipoQ) and its receptors (AdipoRs) are strongly related to growth and development of skeletal muscle, as well as glucose and lipid metabolism in vertebrates. Herein we report the identification of the first full-length cDNA encoding an AdipoR homolog (Liv-AdipoR) from the decapod crustacean Litopenaeus vannamei using a combination of next generation sequencing (NGS) technology and bioinformatics analysis. The full-length Liv-AdipoR (1,245 bp) encoded a protein that exhibited the canonical seven transmembrane domains (7TMs) and the inversed topology that characterize members of the progestin and adipoQ receptor (PAQR) family. Based on the obtained sequence information, only a single orthologous AdipoR gene appears to exist in arthropods, whereas two paralogs, AdipoR1 and AdipoR2, have evolved in vertebrates. Transcriptional analysis suggested that the single Liv-AdipoR gene appears to serve the functions of two mammalian AdipoRs. At 72 h after injection of 50 pmol Liv-AdipoR dsRNA (340 bp) into L. vannamei thoracic muscle and deep abdominal muscle, transcription levels of Liv-AdipoR decreased by 93% and 97%, respectively. This confirmed optimal conditions for RNAi of Liv-AdipoR. Knockdown of Liv-AdipoR resulted in significant changes in the plasma levels of ammonia, 3-methylhistine, and ornithine, but not plasma glucose, suggesting that that Liv-AdipoR is important for maintaining muscle fibers. The chronic effect of Liv-AdipoR dsRNA injection was increased mortality. Transcriptomic analysis showed that 804 contigs were upregulated and 212 contigs were downregulated by the knockdown of Liv-AdipoR in deep abdominal muscle. The significantly upregulated genes were categorized as four main functional groups: RNA-editing and transcriptional regulators, molecular chaperones, metabolic regulators, and channel proteins.

PAQR3 plays a suppressive role in laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a common type of head and neck malignancy worldwide. The molecular mechanism of LSCC remains not well understood. PAQR3 (progestin and AdipoQ receptors) is a member of the progestin and adipoQ receptor (PAQR) family. Many studies have showed that PAQR3 acts as a new tumor suppressor. However, its expression and functions in LSCC are still unknown. In our study, the expression of PAQR3 was downregulated in human LSCC tissues compared to that in adjacent tissues. Moreover, overexpression of PAQR3 suppressed LSCC cell proliferation and invasion. In addition, PAQR3 inhibited cell proliferation and invasion by regulating ERK phosphorylation. In conclusion, our data demonstrated that PAQR3 acted a tumor suppressive role in LSCC, providing a novel diagnostic and therapeutic option for LSCC in the future.

Role of RKTG, a novel Golgi localized receptor, in Gβγ regulated transport of secretory proteins from the Golgi to the plasma membrane (604.4)

Recent studies have revealed a role for G protein betagamma signaling at the Golgi via protein kinase D (PKD) regulating fission of plasma membrane (PM) destined secretory vesicles from the trans Golgi network (TGN). The mechanism by which the Gbetagamma subunits are recruited to and activated at the Golgi in this process remains elusive. Recent studies reveal that a newly characterized Golgi localized, membrane protein belonging to the Progestin and AdipoQ Receptor (PAQR) family, RKTG (Raf Kinase Trapping to the Golgi), interacts with the Gbeta subunit and sequesters Gbetagamma to the Golgi thereby regulating the functions of Gbetagamma. Here we show that over expression of RKTG causes vesiculation of the Golgi, while a Gbeta binding mutant of RKTG does not cause vesiculation. Also, the C‐terminal fragment of GRK2 (GRK2ct), which interacts with, and inhibits Gbetagamma signaling, and Gallein, a small molecule inhibitor of Gbetagamma, were both able to inhibit Golgi vesiculation in the presence of RKTG. Furthermore, a dominant negative form of PKD (PKD‐DN) and a pharmacological inhibitor of PKD, Go6976, also inhibited RKTG mediated vesiculation of the Golgi. Collectively, these results reveal a novel role for the newly characterized, Golgi localized RKTG receptor, in regulating Gbetagamma at the Golgi, thus controlling Golgi to PM protein transport via the Gbetagamma‐PKD signaling pathway.Grant Funding Source: NIH GM056444

Non-genomic rapid responses via progesterone in human peripheral T cells are not indirectly mimicked by sphingosine 1-phosphate

Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. Progesterone has been shown to induce rapid responses that cause intracellular calcium ([Ca2+]i) elevation and acidification followed by inhibition of phytohemagglutinin (PHA)-stimulated proliferation. These rapid responses involve T cell plasma membrane sites, but the mechanisms remain unclear. Three new membrane progesterone receptors (mPRα/mPRβ/mPRγ) have been identified as expressed in T cells. These proteins have been identified as G-protein-coupled receptors. Recently, mPRs have been classified as progestin and adipoQ receptors (PAQRs). Furthermore, they have been suggested to be alkaline ceramidases, possibly involved in mediating sphingolipid signaling. Alkaline ceramidases are capable of converting ceramide to sphingosine, which might then be further phosphorylated sphingosine via sphingosine kinase to sphingosine 1-phosphate (S1P). This pathway could result in progesterone acting indirectly via S1P on membrane sphingosine 1-phosphate receptors (S1PRs) in T cells to induce rapid responses. Therefore, our aim was to investigate whether progesterone rapid responses occur indirectly in T cells via S1P. We found that S1P induces [Ca2+]i elevation however there was no change in intracellular pH. This is different from the situation with progesterone: S1P alone does not suppress PHA-stimulated cell proliferation and does not act synergistically with progesterone on the inhibition of PHA-induced cell proliferation. In contrast, S1P at 1μM is able to antagonize the proliferation inhibitory effect of progesterone. Thus the rapid responses that are induced by progesterone in human peripheral T cells probably do not involve indirect signaling via S1P and S1PRs.