Retinoic acid affects gene expression and morphogenesis without upregulating the retinoic acid receptor in the ascidian Ciona intestinalis

Many chordate-specific morphological features develop depending on retinoic acid (RA). We isolated cDNA clones encoding a retinoic acid receptor (CiRAR) and a retinoid X receptor (CiRXR) in the ascidian Ciona intestinalis. CiRAR mRNA was detected in the anterior ectoderm and endoderm during gastrulation. The expression persists in the head endoderm and two discrete regions of the nerve cord in the tailbud embryo. CiRXR mRNA was ubiquitously expressed. RA affected closure of the neural tube and formation of the adhesive papillae. However, no obvious upregulation in CiRAR expression was observed. Expression of some, but not all, of the neural and papilla-specific genes was reduced in the RA-treated embryo. These results suggest limited roles of CiRAR in ascidian embryos.     

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.     

Functional domains of the gonadotropin-releasing hormone receptor

Summary 1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation.2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor.3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

Steroid receptor coactivator-3 is required for progesterone receptor trans-activation of target genes in response to gonadotropin-releasing hormone treatment of pituitary cells

Regulation of gonadotropin production involves interplay between steroids and neuropeptides, and we have examined the effects of gonadotropin-releasing hormones (GnRH I and GnRH II) on progesterone receptor (PR) activation in alphaT3-1 pituitary cells. Treatment with GnRHs activated a progester-one response element (PRE)-luciferase reporter gene, and this was blocked by protein kinase C and protein kinase A inhibitors but not by RU486. Treatment with GnRHs phosphorylated the PR at Ser(294) and increased PR translocation to the nucleus within 1 h. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR-steroid receptor coactivator-3 (SRC-3) interactions within 8 h. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 by the PREs of the luciferase reporter gene or the gonadotopin alpha-subunit gene promoter was also increased by GnRHs within 8 h, while progesterone-induced recruitment of PR to the PREs occurred in association with much less SRC-3. A small interfering RNA knockdown of type I GnRH receptor levels reduced PR activation by GnRHs, while progesterone-dependent PR activation was unaffected. Moreover, small interfering RNA knockdown of SRC-3 abolished PRE-luciferase trans-activation by the PR in response to GnRHs. Collectively, these data indicate that PR activation by GnRHs in alphaT3-1 cells is type I GnRH receptor-mediated and that trans-activation of PR-responsive genes requires SRC-3 in this context.     

Mapping of gonadotropin-releasing hormone receptor binding site

On the basis of the spatial conformation of gonadotropin-releasing hormone (GnRH), we have predicted that aromatic amino acids and at least one carboxyl group are involved in the recognition site of the receptor. Therefore, various specific reagents were examined for their ability to interfere with the binding of GnRH to its receptor. Pretreatment of pituitary membrane preparations with sodium periodate decreased the specific binding in a dose-dependent manner (IC50 = 0.5 mM) due to a decrease in receptor affinity. This indicated the presence of a sugar moiety in the binding site. Tryptophan is another constituent that participates in the GnRH binding site, as pretreatment of pituitary membranes with 2-methoxy-5-nitrobenzyl bromide inhibited the binding (IC50 = 0.22 mM) by decreasing receptor affinity. In addition, the native hormone conferred on the binding site a protective effect against inactivation by 2-methoxy-5-nitrobenzyl bromide. Pretreatment of membranes with p-diazobenzenesulfonic acid also inhibited the binding of 125I-Buserelin (IC50 = 0.1 mM), indicating the presence of tyrosine within or near the binding site. Pretreatment of pituitary membrane preparations with dithiothreitol also inhibited the binding due to a decrease in the binding affinity, which was accompanied by an increase in receptor number. These data suggest that there are disulfide bonds within or near the binding region. Treatment with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and glycine ethyl ester also prevented binding in a dose-dependent manner and implies that free carboxylic groups are involved in the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)