Cloning and characterization of a family C orphan G-protein coupled receptor

Members of the family C receptors within the G-protein coupled receptor superfamily include the metabotropic glutamate receptors, GABA(B) receptors, the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and a small group of uncharacterized orphan receptors. We have cloned and studied the mouse GPRC6A family C orphan receptor. The open reading frame codes for a protein with highest sequence identity to the fish 5.24 odorant receptor and the mammalian CaSR. The gene structure shows a striking resemblance to that of the CaSR. Results from RT-PCR analyses showed that mouse GPRC6A mRNA is expressed in mouse brain, skeletal muscle, heart, lung, spleen, kidney, liver, and in the early stage mouse embryo. Immunocytochemical analysis of the cloned mouse GPRC6A cDNA expressed in human embryonic kidney 293 cells demonstrated that GPRC6A was present on the plasma membrane, as well as in the endoplasmic reticulum and nuclear envelope membranes of transfected cells. A chimeric cDNA construct in which the extracellular ligand binding domain of the fish 5.24 amino acid-activated odorant receptor was ligated to the complementary downstream sequence of the mouse GPRC6A receptor indicated that GPRC6A is coupled to phosphoinositol turnover and release of intracellular calcium. Further studies with mouse GPRC6A expressed in Xenopus laevis oocytes demonstrated that this receptor possesses a pharmacological profile resembling that of the fish 5.24 odorant receptor. These findings suggest that GPRC6A may function as the receptor component of a novel cellular transmitter system in mammals.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

孤儿受体TR3在小鼠睾丸中的定位和表达

本文采用原位杂交和免疫组织化学技术,观察孤儿受体ＴＲ３及其ｍＲＮＡ在小鼠睾丸中的表达及细胞定位。结果表明,在小鼠睾丸中有显著量的孤儿受体ＴＲ３ ｍＲＮＡ和蛋白表达,其表达量在不同曲细精管有明显的差异;孤儿受体ＴＲ３蛋白主要定位于生精细胞,其ｍＲＮＡ在生精细胞特异表达,主要在精原细胞和发育早期的初级精母细胞表达,提示孤儿受体ＴＲ３在小鼠曲细精管精子发生的早期阶段中起着调控作用。     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Molecular cloning and characterization of the germ cell‐related nuclear orphan receptor in chickens

Nuclear receptor subfamily 6, group A, member 1 (NR6A1), also known as germ cell nuclear factor/retinoid receptor‐related testis‐associated receptor and neuronal cell nuclear factor, is a member of the nuclear orphan receptor superfamily. NR6A1 has been cloned in various species including humans and mice, but it has been scarcely investigated in avian species. In the present study, we cloned the chicken NR6A1 (cNR6A1) from a testis cDNA library. The cloned cNR6A1 sequence was mapped to chromosome 17 and contained an open reading frame of 1.4 kb encoding 445 amino acids. Multiple alignment analysis of the cNR6A1 protein‐coding sequence with NR6A1s from humans, mice, boars, rats, zebrafish, and Xenopus showed high degrees of homology, 89%, 90%, 89%, 88%, 83%, and 87%, respectively. Using RNA interference, changes in the expression of pluripotency‐, germ cell‐, and differentiation‐related key genes by silencing of cNR6A1 were validated in chicken blastoderm‐derived embryonic stem cells. Among those genes, the relative expression levels of POU5F1, CRIPTO, DAZL, DDX4, BMP15, GSC, and SOX7 changed significantly compared to the control group. We also confirmed that the activity of alkaline phosphatase, known as a pluripotency marker, was maintained by cNR6A1 gene silencing in chicken blastodermal cells. Collectively, our data suggest that cNR6A1 may play an important role during chicken embryonic development and differentiation. Mol. Reprod. Dev. 77: 273–284, 2010. © 2009 Wiley‐Liss, Inc.     

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 ^?9 and 10 ^?7M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.     

Morphology of the X-irradiated testes of Dermestes frischii Kugelann (Coleoptera: Dermestidae). I. Somatic cells and pre-spermatid germ cells

During the development of a sterile male control method for Dermestes frischii Kugelann, testis follicles exposed to an X-ray dose of 3.0 krad were investigated using light and electron microscopy.

There was considerable variation in the radiation sensitivity of the various somatic and germ cells. The cyst wall cells seemed particularly resistant while the inner layer of the bilayered follicle sheath was destroyed. The general resistance and versatility of the outer sheath layer maintained the integrity of the follicles. The only outer sheath and apical cells observed to decay were those in close proximity to degenerating germ cells. In some follicles all primary spermatogonia were destroyed, in others their numbers were only depleted. The surviving cells all underwent mitotic delay for 7–14 days. Their subsequent offspring were frequently found to break down and sometimes were proliferated so that germarial polarity was lost. All secondary spermatogonia but only a few primary spermatocytes were destroyed. The decay of germ cells within the same cyst did not necessarily proceed synchronously.     

Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells

In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic-sensors which can up-regulate the functional expression of drug transporters, such as P-glycoprotein (P-gp). In the brain, an increase in P-gp expression can further limit drug permeability across the blood-brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P-gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand-induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small-interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.     

Cloning of a novel neuronally expressed orphan G-protein-coupled receptor which is up-regulated by erythropoietin, interacts with microtubule-associated protein 1b and colocalizes with the 5-hydroxytryptamine 2a receptor

G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides. It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome. Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene. This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs. The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks. A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b. Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor. Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor. The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged. We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.     

Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes

Recently cDNA encoding the histamine H3 receptor was isolated after 15 years of considerable research. However, several studies have proposed heterogeneity of the H3 receptor. We report here the molecular cloning and characterization of a novel type of histamine receptor. A novel orphan G-protein-coupled receptor named GPRv53 was obtained through a search of the human genomic DNA data base and analyzed by rapid amplification of cDNA ends (RACE). GPRv53 possessed the features of biologic amine receptors and had the highest homology with H3 receptor among known G-protein-coupled receptors. Mammalian cells expressing GPRv53 were demonstrated to bind and respond to histamine in a concentration-dependent manner. In functional assays, not only an H3 receptor agonist, R-(alpha)-methylhistamine, but also a H3 receptor antagonist, clobenpropit, and a neuroleptic, clozapine, activated GPRv53-expressing cells. Tissue distribution analysis revealed that expression of GPRv53 is localized in the peripheral blood leukocytes, spleen, thymus, and colon, which was totally different from the H3 receptor, whose expression was restricted to the brain. The discovery of the GPRv53 receptor will open a new phase of research on the physiological role of histamine.