Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.

A series of small compounds acting at the orphan G proteincoupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G q/11 -and G s -mediated signaling pathways, whereas NAG activated only the G q/11 -mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr 97 , Gly 98 , Phe 101 , and Arg 267 of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca 2؉ levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

clones were isolated from human and mouse genomic libraries. The SHP gene was composed of two exons interrupted by a single intron spanning approximately 1.8 kilobases in human and 1.2 kilobases in mouse. Genomic Southern blot analysis and fluorescence in situ hybridization of human metaphase chromosomes indicated that the SHP gene is located at the human chromosome 1p36.1 subband. The 5-flanking regions of human and mouse SHP genes were highly conserved, showing 77% homology in the region of approximately 600 nucleotides upstream from the transcription start site. Primer extension analysis was carried out to determine the transcription start site of human SHP to 32 nucleotides downstream of a potential TATA box. The human SHP gene was specifically expressed in fetal liver, fetal adrenal gland, adult spleen, and adult small intestine. As expected from this expression pattern, the activity of the mouse SHP promoter measured by transient transfection was significantly higher in the adrenal-derived Y1 cells than HeLa cells.The nuclear receptor superfamily is a group of transcription factors regulated by small hydrophobic hormones such as retinoic acid, thyroid hormone, and steroids and also includes a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (for reviews see Refs. 1, 2). The nuclear receptors directly regulate transcription by binding to specific DNA sequences named hormone response elements, generally located in promoters of target genes. The nuclear hormone receptors share a common domain structure. The central DNA binding domain (DBD) 1 includes two zinc binding modules, which consist of a series of invariant cysteine residues. A conserved helical region termed the P box within the DBD (3) makes base-specific contacts and serves as one of the main criteria used for classification of the nuclear receptor superfamily. The C-terminal ligand binding domain (LBD) binds to the cognate ligands. This domain also contains dimerization and transcriptional activation functions. A less well conserved hinge domain that separates DBD and the ligand binding domain has been thought to serve merely as a flexible linker. However, recent results demonstrate that it is also involved with transcriptional repression, at least for a subset of receptors (4). In addition, it was also shown to contain nuclear localization signals (1, 2). A quite variable N-terminal domain includes a transcriptional activation function with some receptors.Although ligands have not been identified for orphan nuclear receptors, a variety of results indicate that they have important functions. The simplest is that knockout mutations of these orphans in mice frequently have shown much more dramatic defects than similar mutations of the conventional receptor genes (5-7). We have recently reported an unusual orphan member of the nuclear receptors that contains a ligand binding domain but lacks the conserved DBD (8). This orphan receptor interacts, both in vitro and in the yeast two-hybrid system wi...

The organization of DNA in chromatin is involved in repressing basal transcription of a number of inducible genes. Biochemically defined multiprotein complexes such as SWI/SNF (J. Côté, J. Quinn, J. L. Workman, and C. L. Peterson, Science 265:53-60, 1994) and nucleosome remodeling factor (T. Tsukiyama and C. Wu, Cell 83:1011-1020, 1995) disrupt nucleosomes in vitro and are thus candidates for complexes which cause chromatin decondensation during gene induction. In this study we show that the glucocorticoid receptor (GR), a hormone-inducible transcription factor, stimulates the nucleosome-disrupting activity of the SWI/SNF complex partially purified either from HeLa cells or from rat liver tissue. This GR-mediated stimulation of SWI/SNF nucleosome disruption depended on the presence of a glucocorticoid response element. The in vitro-reconstituted nucleosome probes used in these experiments harbored 95 bp of synthetic DNA-bending sequence in order to rotationally position the DNA. The GR-dependent stimulation of SWI/SNF-mediated nucleosome disruption, as evaluated by DNase I footprinting, was 2.7-to 3.8-fold for the human SWI/SNF complex and 2.5-to 3.2-fold for the rat SWI/SNF complex. When nuclear factor 1 (NF1) was used instead of GR, there was no stimulation of SWI/SNF activity in the presence of a mononucleosome containing an NF1 binding site. On the other hand, the SWI/SNF nucleosome disruption activity increased the access of NF1 for its nucleosomal binding site. No such effect was seen on binding of GR to its response element. Our results suggest that GR, but not NF1, is able to target the nucleosome-disrupting activity of the SWI/SNF complex.DNA in eukaryotic cells associates with proteins called histones to form nucleosomes, which together with nonhistone proteins form a higher-order structure, chromatin (22). It is now well established that chromatin not only provides a DNA storage function but is also involved in gene regulation (41,63). When the synthesis of histone H4 is inhibited in yeast cells, several genes which are tightly regulated are expressed in a constitutive manner (14). The mouse mammary tumor virus (MMTV) promoter, which is repressed in the absence of glucocorticoid hormone, becomes constitutively expressed when the nucleosome density is decreased by coinjection of competitor DNA, as shown in Xenopus oocytes (43). Injection of single-stranded DNA into Xenopus oocytes leads to nucleosome assembly coupled to DNA synthesis. This results in a tighter chromatin structure which confers a more stringent repression of transcription than chromatin formed on DNA injected in the double-stranded form (2). Several studies suggest that chromatin acts both by excluding certain upstreamgene-specific transcription factors from their recognition sites (1, 4) and by inhibiting access of the basic transcription machinery to the transcriptional initiation site (17,30,33,64).Regulatory regions of many inducible genes in mammals and yeast have positioned nucleosomes. Examples include the MMTV promoter (53), the...

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