Ute Süsens

The Germ Cell Nuclear Factor mGCNF Is Expressed in the Developing Nervous System

Using a reinoic acid receptor hybridization probe, we have isolated a mouse embryonic cDNA that encodes the germ cell nuclear factor (mGCNF). The in vitro translated protein binds specifically to the direct repeat of the sequence-AGGTCA-which is characteristic of a subclass of nuclear receptors, albeit with a spacing of zero that is unique among the receptors. Northern analysis shows embryonic expression after mouse gastrulation and during early organogenesis, as well as expression in the adult testis. The message level decreases during embryonic development. Whereas there are two transcripts in the testis, only the larger one is found in embryos. In situ analysis of embryos at days 6.5-10.5 of gestation shows that, in the early postimplantation period, high transcript levels are found in ectodermal cells and in the primitive streak. During further development the expression becomes more restricted. Mainly cells of the developing nervous system are expressing the receptor. Our findings imply that GCNF is involved in two apparently disparate developmental events.

Unique expression pattern of a novel mosaic receptor in the developing cerebral cortex

Recently, a new type of transmembrane protein with a unique combination of protein domains was characterized from human, rabbit and chicken. This protein exhibits features of the low-density lipoprotein receptor family and shows homology to the receptor of the neuropeptide head activator isolated from hydra. To study the temporal and spatial pattern of expression of this unusual new receptor we have isolated a murine homolog and, in accordance with its human counterpart, named it mSorLA. Northern blot analysis revealed the highest abundance of mSorLA transcripts in the adult brain, lower levels in a variety of other organs and expression during embryogenesis. In situ hybridization showed predominant localization in neurons of the cortex, the hippocampus and the cerebellum. During embryonic development mSorLA displayed a unique pattern of expression in the cerebral cortex, where a subpopulation of neurons was labeled before final differentiation. Transcripts of mSorLA were also detected outside the central nervous system in regions active in morphogenesis.

PGC-1 and PERC, coactivators of the estrogen receptor-related receptor γ

The mouse nuclear receptor ERRgamma (estrogen receptor-related receptor gamma) is highly expressed in heart, skeletal muscle, kidney, and brain, as well as in the developing nervous system. We found that the expression of the coactivators PGC-1 (PGC-1alpha) and PERC (PGC-1beta) in mammalian cells augmented potently the transcriptional activation by ERRgamma. The constitutive activation function 2 (AF-2) of the orphan receptor was important for the synergistic enhancement. Functional receptor truncation analysis revealed an additional amino-terminal activation function, specific for the ERRgamma2 isoform and PGC-1. In vitro experiments showed a direct interaction of ERRgamma with both coactivators. Our findings suggest distinct regulatory functions for PGC-1 and PERC as tissue-specific coactivators for ERRgamma.

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

The neuropeptide head activator (HA) is a mitogen for mammalian cell lines of neuronal or neuroendocrine origin. HA signalling is mediated by a G-protein-coupled receptor (GPCR). Orphan GPCRs with homology to peptide receptors were screened for HA interaction. Electrophysiological recordings in frog oocytes and in mammalian cell lines as well as Ca2+ mobilisation assays revealed nanomolar affinities of HA to GPR37. HA signal transduction through GPR37 was mediated by an inhibitory G protein and required Ca2+ influx through a channel of the transient receptor potential (TRP) family. It also required activation of Ca2+-dependent calmodulin kinase and phosphoinositide 3-kinase. Respective inhibitors blocked HA signalling and HA-induced mitosis in GPR37-expressing cells. HA treatment resulted in internalisation of GPR37. Overexpression of GPR37 led to aggregate formation, retention of the receptor in the cytoplasm and low survival rates of transfected cells, confirming the notion that misfolded GPR37 contributes to cell death, as observed in Parkinsons disease.

Differential expression of the estrogen receptor-related receptor γ in the mouse brain

To elucidate estrogen functions, the expression of the estrogen receptor-related receptor ERRγ, a novel orphan nuclear receptor regulating transcription via estrogen responsive elements, has been localized by in situ hybridization in adult murine brain. ERRγ transcripts were abundantly present in the isocortex, the olfactory system, cranial nerve nuclei and major parts of the coordination centers, e.g. reticular formation and major parts of the extrapyramidal motor systems. In addition, ERRγ expression was detected in trigeminal ganglion neurons. ERRγ distribution was clearly distinguished from that described for ERRα, for ERRβ, and for estrogen receptors (ER) pointing at functional differences between ERRγ and these receptors.

Retinoids Induce Differential Expression and DNA Binding of the Mouse Germ Cell Nuclear Factor in P19 Embryonal Carcinoma Cells

The mouse germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily, is highly expressed during gametogenesis and in the developing nervous system. The in vitro translated protein binds as a homodimer to the direct repeat (DR) of the sequence -AGGTCA- (DR-0). In this report, we characterize a DR-0 binding activity in P19 cell extracts that is induced by retinoids. This induction is concentration dependent and specific for embryonal carcinoma cells. The cellular protein binds with the same specificity as in vitro expressed GCNF, but migrates as a slower complex, indicating interaction with partner proteins. Because antisera directed against GCNF recognize this complex, we propose that GCNF is part of the binding activity. Combining in vitro translated GCNF and extracts of non-expressing cells shows that such interactions can be formed posttranslationally. Northern analysis demonstrates a concentration dependent induction of GCNF mRNA by retinoic acid. A time course shows that the level of GCNF binding is transiently elevated, later downregulated, and not detectable in differentiated cells. We propose that GCNF regulation is an important step during determination of embryonal carcinoma cells.

Characterization of the human germ cell nuclear factor gene

A cDNA clone encoding the germ cell nuclear factor, GCNF, a member of the nuclear receptor superfamily has been isolated from the human embryonal carcinoma cell line NT2/D1. Sequencing of this clone reveals an open reading frame encoding a 476 amino acid protein. A comparison of the amino acid sequence of the human GCNF with its mouse homologue shows only six amino acid exchanges in the whole protein and a deletion in the amino-terminal region. Northern blot analysis demonstrates that the expression in the testis is conserved.

Developmental expression of the estrogen receptor-related receptor γ in the nervous system during mouse embryogenesis

The ERRs (estrogen receptor-related receptors) are constitutive activators of the classical estrogen response element. In this report, we demonstrate that ERRgamma is highly expressed in the nervous system of the developing mouse embryo and that the adult pattern of expression of ERRgamma is, with few exceptions, established during embryogenesis. Transcripts are preferentially detected in already differentiating areas of the nervous system.

Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.

By searching the human and mouse genomic databases we found two G-protein-coupled receptors, GPR139 and GPR142, with characteristic motifs of the rhodopsin family of receptors. The gene for GPR139 maps to chromosome 7F1 of mouse and 16p12.3 of human and that for GPR142 to 11E2 of mouse and 17q25.1 of human. We isolated GPR139 from a cDNA library of adult mouse brain and GPR142 from a cDNA library of brains from 15-day-old mouse embryos. GPR139 mRNA was predominantly expressed in specific areas of human and mouse brains, whereas GPR142 mRNA showed a more ubiquitous expression both in the brain and in various peripheral glands and organs. A 50% identity and a 67% homology at the amino-acid level between the two receptors and only 20-25% identity with other G-protein-coupled receptors established them as a new subbranch within the phylogenetic tree and hints at a common or similar ligand(s). Preliminary results suggest that the cognate ligand is present in brain extracts and is, most likely, a small peptide. GPR139 signal transduction in Chinese hamster ovary cells requires coupling to an inhibitory G-protein and is mediated by phospholipase C. Dimer formation may be necessary for proper function.

DNA binding, protein interaction and differential expression of the human germ cell nuclear factor

The mouse germ cell nuclear factor (mGCNF) is an orphan nuclear receptor implicated in diverse biological processes, including gametogenesis, embryonic development and embryonal carcinoma cell differentiation. We have examined the binding and regulation of the human orthologue, hGCNF, expressed in the teratocarcinoma-derived cell line NTera-2/clone D1 (NT2/D1). Binding of GCNF to the direct repeat of the sequence -AGGTCA- (DR-0) is conserved in mammalia. The formation of interspecies dimers of the in vitro synthesized proteins suggests that cellular GCNF binding is mediated by homodimers. Both the mouse and the human protein bind in concert with cellular factors to DNA. Treatment of NT2/D1 cells with all-trans retinoic acid (atRA) is accompanied first by an up-regulation followed later by a down-regulation of hGCNF and its mRNA. Temporary up-regulation in NT2/D1 cells after treatment with atRA suggests that hGCNF is important for human neural determination and differentiation.