Search

Diuresis following blood-gorging in Rhodnius prolixus is the major process leading to the transmission of Chagas' disease. We have cloned the cDNA of the first receptor known to be involved in an antidiuretic strategy in insects, a strategy that prevents diuresis. This receptor belongs to the insect CAPA receptor family known in other insects to be activated by peptides encoded within the capability gene. We characterize the expression profile in fifth-instars and find expression is localized to the alimentary canal. Highest transcript levels are found in Malpighian tubules and the anterior midgut, which are known targets of the antidiuretic hormone, RhoprCAPA-[alpha]2. Two transcripts were identified, capa-r1 and capa-r2; however, the latter encodes an atypical G protein-coupled receptor lacking a region ranging between the first and second transmembrane domain. Our heterologous expression assay revealed the expressed capa-r1 receptor is activated by RhoprCAPA-[alpha]2 ([EC.sub.50] = 385nM) but not by RhoprCAPA-[alpha]1. Structural analogs of the inactive RhoprCAPA-[alpha]1 were capable of activating the expressed capa-r1 receptor, confirming the importance of the C-terminal consensus sequence common to CAPA-related peptides. In addition, this receptor has some sensitivity to the pyrokininrelated peptide, RhoprCAPA-[alpha]PK1, but with an efficacy [approximately equal to] 40-fold less than RhoprCAPA-[alpha]2. Other peptides belonging to the PRXa-mide superfamily were inactive on the capa-r1 receptor. Taken together, the neuroendocrinological relevance of this receptor in facilitating the antidiuretic strategy in R. prolixus may make this receptor a useful target for development of agonists or antagonists that could help influence the transmission of Chagas' disease that occurs during diuresis in this medically important insect-disease vector. CAPA | Chagas' | G protein-coupled receptor | neurohormone | neuropeptide doi/ 10.1073/pnas.1003666107

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1(CRFR1) on primary [beta] cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erkl/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal [beta] cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFRl-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on [beta] cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate [beta] cell proliferation. diabetes | pancreas | corticotropin-releasing factor | hypothalamus-pituitary-adrenal axis | glucose homeostasis www.pnas.org/cgi/doi/10.1073/pnas.0913610107

We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene--deleted mice ([sst.sub.4.sup.-/-]), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease models; several symptoms are more severe in the [sst.sub.4] knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction in the skin are much greater in mice lacking the [sst.sub.4] receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration are also markedly enhanced in [sst.sub.4] knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet cell hyperplasia, myeloperoxidase activity, and 1L-1[beta], TNF-[alpha], and IFN-[gamma] expression in the inflamed lung. It is concluded that during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through [sst.sub.4] receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic drugs. allergic contact dermatitis | arthritis | capsaicin-sensitive afferents | endotoxin-induced pneumonitis | inflammatory cytokines

Glucocorticoid receptor [alpha] (GR[alpha]) and peroxisome proliferator-activated receptor [alpha] (PPAR[alpha]) are transcription factors with clinically important immune-modulating properties. Either receptor can inhibit cytokine gene expression, mainly through interference with nuclear factor [kappa]B (NF-[kappa]B)-driven gene expression. The present work aimed to investigate a functional cross-talk between PPAR[alpha]- and GR[alpha]-mediated signaling pathways. Simultaneous activation of PPAR[alpha] and GR[alpha] dose-dependently enhances transrepression of NF-[kappa]B-driven gene expression and additively represses cytokine production. In sharp contrast and quite unexpectedly, PPAR[alpha] agonists inhibit the expression of classical glucocorticoid response element (GRE)-driven genes in a PPAR[alpha]-dependent manner, as demonstrated by experiments using PPAR[alpha] wild-type and knockout mice. The unclerlying mechanism for this transcriptional antagonism relies on a PPAR[alpha]-mediated interference with the recruitment of GR[alpha], and concomitantly of RNA polymerase II, to GRE-driven gene promoters. Finally, the biological relevance of this phenomenon is underscored by the observation that treatment with the PPAR[alpha] agonist fenofibrate prevents glucocorticoid-induced hyperinsulinemia of mice fed a high-fat diet. Taken together, PPAR[alpha] negatively interferes with GRE-mediated GR[alpha] activity while potentiating its antiinflammatory effects, thus providing a rationale for combination therapy in chronic inflammatory disorders. cross-talk | gluconeogenesis | inflammation | hyperinsulinema | side effects

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1-29)N[H.sub.2] (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of [sup.125]l-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1-42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control. Ghrelin | Ghrelin receptor | GHRH

Transcriptional regulators such as the glucocorticoid receptor (GR) recruit multiple cofactors to activate or repress transcription. Although most cofactors are intrinsically bifunctional, little is known about the molecular mechanisms dictating the specific polarity of regulation. Furthermore, chromatin modifications thought to be confined to silent loci appear in actively transcribed genes suggesting that similar enzymatic activities may mediate constitutive and transient chromatin states. GRIP1, a GR ligand-dependent coregulator of the p160 family can potentiate or inhibit transcription but the molecular contexts and mechanisms that enable GRIP1 corepressor activity are poorly understood. In a yeast 2-hybrid screen with GRIP1 repression domain (RD)-containing fragment, we repeatedly isolated the C-terminal region of a SET domain-containing protein subsequently identified as histone H4 lysine 20 trimethyltransferase, Suv4-20h1. We cloned a full-length Suv4-20h1 and dissected its interaction with GRIP1 in yeast, in vitro, and in mammalian cells. Strict nuclear localization and high salt concentration required for Suv4-20h1 extraction were consistent with its tight association with chromatin. Overexpression of Suv4-20h1 in human U2OS and A549 cells expressing integrated and endogenous GR, respectively, antagonized ligand-dependent induction of a subset of GR target genes, whereas Suv4-20h1 siRNA-mediated depletion had a reciprocal effect. Inhibition of GR transactivation required both the GRIP1 interacting region of Suv4-20h1 and its catalytic activity. Thus, Suv4-20h1 known exclusively as a factor involved in constitutive heterochromatin maintenance, actively participates in hormone-dependent transcriptional regulation affecting GR target gene expression in a promoter- and cell type-specific manner. GRIP1 cofactor | histone methyltransferase | Suv4-20h1 | transcriptional regulation

The thyroid hormone receptor (TR) has been proposed to regulate expression of target genes in the absence of triiodothyronine ([T.sub.3]) through the recruitment of the corepressors, NCoR and SMRT. Thus, NCoR and SMRT may play an essential role in thyroid hormone action, although this has never been tested in vivo. To accomplish this, we developed mice that express in the liver a mutant NCoR protein (L-NCoR[DELTA]ID) that cannot interact with the TR. L-NCoR[DELTA]ID mice appear grossly normal, however, when made hypothyroid the repression of many positively regulated [T.sub.3]-target genes is abrogated, demonstrating that NCoR plays a specific and sufficient role in repression by TR in the absence of [T.sub.3]. Remarkably, in the euthyroid state, expression of many [T.sub.3]-targets is also up-regulated in L-NCoR[DELTA]ID mice, demonstrating that NCoR also determines the magnitude of the response to [T.sub.3] in euthyroid animals. Although positive [T.sub.3] targets were up-regulated in L-NCoR[DELTA]ID mice in the hypo- and euthyroid state, there was little effect seen on negatively regulated [T.sub.3] target genes. Thus, NCoR is a specific regulator of [T.sub.3]-action in vivo and mediates repression by the unliganded TR in hypothyroidism. Furthermore, NCoR appears to play a key role in determining the tissue-specific responses to similar levels of circulating [T.sub.3]. Interestingly, NCoR recruitment to LXR is also impaired in this model, leading to activation of LXR-target genes, further demonstrating that NCoR recruitment regulates multiple nuclear receptor signaling pathways. gene expression | thyroid hormone receptor

Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding 'hot-spot' consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its coregulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-activated transcription factor that mediates the stimulatory effects of estrogens and the inhibitory effects of antiestrogens such as tamoxifen in breast cancer and other estrogen target cells. To understand how antiestrogens and dominant negative ERs suppress ER activity, we used a dominant negative ER as bait in two-hybrid screening assays from which we isolated a clone from breast cancer cells that potentiates the inhibitory activities of dominant negative ERs and antiestrogen-liganded ER. At higher concentrations, it also represses the transcriptional activity of the estradiol-liganded ER, while having no effect on other nuclear hormone receptors. This clone, denoted REA for 'repressor of estrogen receptor activity,' encodes a 37-kDa protein that is an ER-selective coregulator. Its competitive reversal of steroid receptor coactivator 1 enhancement of ER activity and its direct interaction with liganded ER suggest that it may play an important role in determining the sensitivity of estrogen target cells, including breast cancer cells, to antiestrogens and estrogens.

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPAR[Gamma] subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPAR[Gamma] ligand that was a weak partial agonist of PPAR[Gamma] transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0-12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12-24 hr, P implant removed; 24-36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3[Alpha]-hydroxy-5[Alpha]-pregnan-20-one, which operates through the type A [Gamma]-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

Although hormone therapy with antiandrogens has been widely used for the treatment of prostate cancer, some antiandrogens may act as androgen receptor (AR) agonists that may result in antiandrogen withdrawal syndrome. The molecular mechanism of this agonist response, however, remains unclear. Using mammalian two-hybrid assay, we report that antiandrogens, hydroxyflutamide, bicalutamide (casodex), cyproterone acetate, and RU58841, and other compounds such as genistein and RU486, can promote the interaction between AR and its coactivator, ARA70, in a dose-dependent manner. The chloramphenicol acetyltransferase assay further demonstrates that these antiandrogens and related compounds significantly enhance the AR transcriptional activity by cotransfection of AR and ARA70 in a 1:3 ratio into human prostate cancer DU145 cells. Our results suggest that the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells. These findings may provide a good model to develop better antiandrogens without agonist activity.

[Cpe.sup.fat] mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in [Cpe.sup.fat] mice. In [Cpe.sup.fat] mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high [K.sup.+] stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of [Cpe.sup.fat] mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of [Cpe.sup.fat] mice.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a 'cross-talk' model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

Environmental perturbations that increase plasma thyroid hormone ([T.sub.3]) concentrations also profoundly affect female reproductive behavior and physiology. We explored whether these effects were mediated by interactions between [T.sub.3] receptor (TR) and estrogen receptor (ER). This hypothesis was of interest because the half-site of a consensus [T.sub.3] response element DNA sequence is identical to an ER response element (ERE), and TRs bind to a consensus ERE. Molecular data presented in the accompanying paper [Zhu, Y.-S., Yen, P.M., Chin, W. W. & Pfaff, D. W. (1996) Proc. Natl. Acad. Sci. USA 93, 12587-12592] demonstrate that TRs and ERs are both present in rat hypothalamic nuclear extracts and that both can bind to the promoter the hypothalamic gene preproenkephalin and that interactions between liganded TRs and ERs affect preproenkephalin transcription. In this paper, we show that molecular interactions between TRs and ERs are sufficient to mediate environmental effects on estrogen-controlled reproductive behavior. Ovariectomized (OVX) rats treated with high doses of [T.sub.3] showed significantly lower levels of lordosis behavior in response to estradiol benzoate (EB) compared with OVX females treated with EB alone. Conversely, thyroidectomized/OVX females treated with EB showed significantly greater levels of lordosis behavior compared with OVX females treated with EB, showing the effect of endogenous [T.sub.3]. Thyroid hormone interference with EB-induced behavior could not be explained by a reduction in plasma [E.sub.2] concentrations or by a general reduction in responsiveness of EB-sensitive tissues. Moreover, numbers of hypothalamic ER-immunoreactive cells increased dramatically following [T.sub.3] treatment. These data suggest that [T.sub.3] may reduce EB-dependent sexual behavior through interactions between TR and ER in the nuclei of behaviorally relevant hypothalamic neurons, envisioning for the first time a functional consequence of interactions between two nuclear hormone receptors in brain. These results also open up the possibility of molecular interactions on DNA encoding environmental signals, a new field for the study of neuronal integration.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal [beta]-oxidation system enzymes by activating peroxisome some proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone ([T.sub.3]; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and [T.sub.3] signaling pathways. [T.sub.3] inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal [beta]-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and [T.sub.3] signaling pathways occurs through their common with the heterodimeric partner RXR.

Analysis of genomic DNA from 32 unrelated families with male-limit precocious puberty subjected to Asp578 to Gly, Met571 to Ile and Thr577 to Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR) shows that heterogeneous point mutations occur mainly in the region covering nt 1624-1741 of exon 11. The mutations trigger hLHR to result in male-limited precocious puberty.

Gonadotrophin-releasing hormone receptor agonist down-modulates pertussis toxin-insensitive G proteins G9-alpha and G11-alpha in alpha T3-1 pituitary gonadotrophs. Exposure to the agonist increases the turnover of G9 alpha and G11 alpha but not cause changes in mRNA levels. These results reflect enhanced proteolysis of the activated G proteins.

Coexpression of the cDNA for a member of the nuclear receptor family, ubiquitous receptor (UR), in COS-1 cells prevents the induction of chloramphenicol acetyltransferase (CAT) reporter gene expression by hRXRalpha and human retinoic acid receptor alpha, in the presence of all-trans-retinoic acid. UR, in combination with hRXRalpha, activates DR-4-CAT expression, which is reduced by the coexpression of thyroid hormone receptor. Normal growth and differentiation involve the regulation of gene activation in retinoic acid and thyroid hormone signaling pathways by UR.

Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors which function as transcription factors that control the expression of the acyl-CoA oxidase gene, thus regulating the peroxisomal beta-oxidation of fatty acids. A search for physiological activators of PPAR-alpha showed that retinoic X receptor-beta could bind with PPAR-alpha and the resulting heterodimer stimulates the acyl-CoA oxidase gene promoter. In addition, PPAR-alpha could also be activated by physiological concentrations of fatty acids and a synthetic arachidonic acid analog.

The peptide des-His-1-(Glu-9)glucagon amide is a full glucagon antagonist at receptors on hepatic plasma membranes. The interactions of this antagonist with intact and saponin-permeabilized canine hepatocytes were investigated. The results confirmed that des-His-1-(Glu-9)glucagon amide acts as a glucagon antagonist bybinding to glucagon receptors. Based on the results, a model for the mechanism of action of the glucagon antagonist is presented.

Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-TrpSer-Tyr-Gly-Leu-Arg-Pro-Gly-[NH.sub.2]) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-[NH.sub.2]), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating [K.sup.+] channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to [G.sub.[Alpha]q/11] and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain [poly(A).sup.+] RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 [micro]g of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Only three of the four thyroid hormone receptor (TR) isoforms, [Alpha]1, [Beta]1, and [Beta]2, bind thyroid hormone (TH) and are considered to be true TRs. TR[Alpha]2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TR[Alpha] (TR[[Alpha].sup.o/o]) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine ([T.sub.4]), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-[T.sub.3]) given to TH-deprived and to intact TR[[Alpha].sup.o/o] mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TR[Beta] (TR[[Beta].sup.-/-]). In liver, [T.sub.3] produced significantly greater responses in TR[[Alpha].sup.o/o] and smaller responses in TR[[Beta].sup.-/-] as compared with WT mice. In contrast, cardiac responses to L-[T.sub.3] were absent or reduced in TR[[Alpha].sup.o/o], whereas they were similar in WT and TR[Beta]-/- mice, supporting the notion that TR[Alpha]1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-[T.sub.3]) given to intact mice produced a greater suppression of serum [T.sub.4] in TR[[Alpha].sup.o/o] than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive 'silencing' effect of TR[Alpha]2 in tissues expressing the TR[Beta] isoforms.

The sst2 somatostatin receptor mediates the antiproliferative effects of somatostatin analogs. The present study demonstrates that stable expression of sst2 in the hamster pancreatic cancer cells PC-1 and PC-1.0 activates an autocrine negative loop leading to an in vitro inhibition of cell proliferation. In vivo studies conducted in Syrian golden hamsters after orthotopic implantation of PC-1.0 cells showed that both tumor growth and metastatic progression of allografts containing 100% of sst2-expressing cells were significantly inhibited for up to 20 days after implantation, as compared with control allografts that did not express sst2. A local antitumor bystander effect was observed after induction of mixed tumors containing a 1:3 ratio of sst2-expressing cells to control cells. Tumor volume and incidence of metastases of mixed tumors were significantly reduced at day 13 post implantation. This effect decreased with time as at day 20, growth of mixed tumors was similar to that of control tumors. After administration of the cytotoxic somatostatin conjugate AN-238 on day 13, antitumor bystander effect observed in mixed tumors was significantly extended to day 20. We also observed that in vitro invasiveness of sst2-expressing PC-1.0 cells was significantly reduced. Tyrosine dephosphorylation of E-cadherin may participate in restoring the E-cadherin function, reducing in turn pancreatic cancer cell motility and invasiveness. This dephosphorylation depends on the tyrosine phosphatase src homology 2-containing tyrosine phosphatase I (SHP-1) positively coupled to sst2 receptor. The inhibitory effect of sst2 gene expression on pancreatic cancer growth and invasion combined with chemotherapy with targeted cytotoxic somatostatin analog administration provides a rationale for a therapeutic approach to gene therapy based on in vivo sst2 gene transfer. bystander effect | tumor invasion | targeted chemotherapeutic agent | experimental gene therapy | E-cadherin

The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin-proteasome degradation pathway. Immunoblots demonstrated that the transfected TR[Beta]1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TR[Beta]1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TR[Beta]1 by the proteasome degradation pathway. T3 also induced rapid degradation of TR[Beta]2 and TR[Alpha]1. In contrast, the stability of the non-T3-binding TR[Alpha]2 and naturally occurring TR[Beta]1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TR[Beta]1 protein increased, yet T3-dependent transcriptional activation and the expression of the growth hormone gene were diminished, suggesting that proteasome-mediated degradation played a novel role in modulating transcriptional activation by TR. The present study reveals a role of T3 in modulating the functions of TR by regulating its receptor level via the ubiquitin-proteasome degradation pathway.

Many of the effects of estrogens on the uterus are mediated by ER[Alpha], the predominant ER in the mature organ. Because of the poor reproductive capacity of ER[Beta] knockout (BERKO) female mice (small litter size, multiple-resorbed fetuses), the role of uterine ER[Beta] was explored. In the immature uterus, ER[Alpha] and ER[Beta] are expressed at comparable levels in the epithelium and stroma, and 17[Beta]-estradiol ([E.sub.2]) treatment decreases ER[Beta] in the stroma. The immature uterus of untreated BERKO mice exhibits elevated levels of progesterone receptor (PR) and the proliferation-associated protein, Ki-67. It also exhibits exaggerated responsiveness to [E.sub.2], as indicated by enlargement of the lumen, increase in volume and protein content of uterine secretion, induction of the luminal epithelial secretory protein, complement C3, and its regulatory cytokine IL-1[Beta], and induction of vascular endothelial growth factor and insulin-like growth factor 1 but not its receptor. As expected, [E.sub.2] increased PR in the stroma and decreased it in the luminal epithelium of wild-type mice. In the BERKO uterus, [E.sub.2] induced PR in the stroma but did not down-regulate it in the epithelium. Increased cell proliferation and exaggerated response to [E.sub.2] in BERKO suggest that ER[Beta] plays a role in modulation of the effects of ER[Alpha] and in addition (or as a consequence of this) has an antiproliferative function in the immature uterus.