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Aim: This review focuses on nuclear receptors expressed in pancreatic cancer., Materials and Methods: An extensive search of articles published up to March 2013 was conducted using the MEDLINE database. The key words used were "pancreatic cancer", "molecular receptors" and "growth factors". A total of 112 articles referred to pancreatic cancer, molecular receptors and/or growth factors were included., Results: Receptors of growth factors, such as the epithelial growth factor receptor, insulin-like growth factor-1 receptor, vascular endothelial growth factor receptor and others, such as integrin α5β1, somatostatin receptors, the death receptor 5, claudin, notch receptors, mesothelin receptors, follicle-stimulating hormone receptors, the MUC1 receptor, the adrenomedullin receptor, the farnesoid X receptor, the transferrin receptor, sigma-2 receptors, the chemokine receptor CXCR4, the urokinase plasminogen activator receptor, the ephrine A2 receptor, the GRIA3 receptor, the RON receptor and the angiotensin II receptor AT-1 are expressed in pancreatic tumor cells. These molecules are implicated in tumor growth, apoptosis, angiogenesis, metastasis etc., Conclusion: After identifying the molecular receptors associated with the pancreatic cancer, many more target molecules playing important roles in tumor pathophysiology and senescence-associated signal transduction in cancer cells will be identified. This may have a significant influence on diagnosis, therapy and prognosis of pancreatic cancer., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

AII amacrine cells are critical interneurons in the rod pathway of mammalian retina, active primarily in dim lighting conditions. Melatonin, a neuromodulator produced at night in the retina, is believed to induce retinal adaptation to dim lighting conditions in most vertebrate species examined to date, including humans. We hypothesized that melatonin may influence retinal light adaptation by acting on AII cells directly and thus investigated whether melatonin receptors were expressed in AII neurons. Postmortem nonpathological eyes from four human donors as well as two eyes from two Macaque Fasicularis monkeys were analyzed. Double immunocytochemistry was performed using an anti-MT(1) antibody and an antibody to calretinin, an AII marker. Analysis utilized confocal microscopy. A polyclonal anti-calretinin antibody labelled amacrine cells exhibiting the distinct AII morphology, in both human and macaque retina. MT(1) immunoreactivity in macaque retina was similar to human staining, in that horizontal, amacrine and ganglion cell bodies were stained, as were inner segments of photoreceptors. In human retina 86% of calretinin positive cells expressed the MT(1) receptor peripherally, whereas centrally, 78% colocalization was observed. In the macaque retina, 100% of AII amacrine cells expressed MT(1) immunoreactivity both centrally and peripherally. That virtually all AII neurons express the MT(1) receptor in both human and macaque retina, may provide the first evidence demonstrating a role for melatonin in AII regulation, furthering the hypothesis of melatonin function in retinal light adaptation.

Aim: To examine the distribution of melatonin 1a (MT1) receptors in the human eye., Methods: Seven normal human eyes were examined by immunohistochemical staining of paraffin sections, using an anti-MT1 primary antibody and an ABC detection system., Results: MT1 receptor immunoreactivity (MT1-IR) was detected primarily in the inner segments of rods and cones and in retinal ganglion cells. In addition, MT1-IR was present in the adventitia of retinal arteries and veins, including the papillary region, but absent in ciliary and choroidal vessels. Mild staining of corneal endothelial cells and keratocytes was observed in all but two eyes., Conclusion: MT1-IR is present in various ocular tissues with the highest density in photoreceptor cells and ganglion cells. The physiological function of these receptors deserves further investigation.

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic beta cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G(i/o) coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.

The pineal neurohormone melatonin is widely recognized as exerting important immunoenhancing effects that act on specific receptors in immunocompetent cells. This action results in stimulation of cytokine production in lymphocytes and macrophages. Here we report that the nocturnal plasma concentration of melatonin in rheumatoid arthritis (RA) patients is higher than in healthy controls. Furthermore, melatonin is present in the synovial fluid of RA patients and synovial macrophages express a specific binding site. We suggest that melatonin may exert a disease-promoting role in RA.

Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C-terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt(1338-352)). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N-terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-((125)I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.

Background: The pineal hormone melatonin has been shown to exert a direct oncostatic activity on neoplastic cells, particularly from breast cancer. In the present study, we evaluated the effects of melatonin on the proliferation and on the cell cycle distribution of human androgen-independent DU 145 prostate cancer cells. Experiments were also performed to gain insights into the possible mechanism of action of the hormone., Methods: The effects of melatonin on DU 145 cell proliferation was analyzed by counting the cells by hemocytometer at the end of treatment. The effects of the pineal hormone on cell cycle distribution were evaluated by FACS analysis. RT-PCR studies were performed to detect Mel(1a) and Mel(1b) expression in DU 145 cells. The cellular localization of (125)I-melatonin binding sites was investigated by radioreceptor assay. A commercially available binding-protein assay kit was utilized to evaluate intracellular cAMP levels., Results: Melatonin, in physiological doses, significantly inhibited DU 145 cell proliferation and induced cell cycle withdrawal by accumulating cells in G0/G1 phase. The mRNA for Mel(1a) receptors was found to be expressed in DU 145 cells; however, by radioreceptor assay, no binding sites for (125)I-melatonin could be detected in membrane preparations, suggesting that, in these cells, the level of translation of this mRNA is too low to possibly mediate the antiproliferative action of the hormone. In agreement with this hypothesis, melatonin did not affect forskolin-induced intracellular cAMP accumulation. Binding sites for (125)I-melatonin were found in nuclear extracts of DU 145 cells., Conclusions: Melatonin exerts a direct oncostatic activity on human androgen-independent prostate cancer cells, by affecting cell cycle progression. This activity seems to be mediated by nuclear, but not by membrane, receptors., (Copyright 2000 Wiley-Liss, Inc.)

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

We report the production of polyclonal antibodies directed against the human melatonin receptors Mel-1a (mt1) and Mel-1b (MT2) by means of antigenic synthetic peptides with sequences unique to these proteins. Immunostaining on NIH3T3 cells stably transfected with Mel-1a and Mel-1b cDNA gave intense reactions. Neither the preimmune serum nor cross-tested antisera showed any reactivity. These polyclonal antibodies will be essential immunocytochemical tools to study the human melatonin receptors distribution at subcellular level.

To better define the role of melatonin in fish, we have compared in detail the distribution of 2-[125I]iodomelatonin binding sites with gene expression for melatonin receptor subtypes in a widely studied seasonal species, the rainbow trout. Three distinct partial sequences of the melatonin receptor gene were cloned from trout genomic DNA. Two of the sequences corresponded to the Mella receptor subtype, and one corresponded to the Mellb receptor subtype. Analysis of numerous clones failed to find a sequence equivalent to the Mel1c receptor subtype. Comparison of receptor gene expression with 2-[125I]iodomelatonin binding distribution indicated dendritic transport of the receptor. Melatonin receptors were associated predominantly with visually related areas of the trout brain, such as the thalamic region, the pretectal area, and the optic tectum. The pituitary was devoid of 2-[125I]iodomelatonin binding, and melatonin receptor gene expression was not detectable. It would appear from the results of the present study that melatonin in this species is involved primarily in the processing of visual signals. How melatonin interacts with circannual rhythms of growth and reproduction is unclear, although a direct interaction between melatonin and the hypothalamo-pituitary axis is not clearly indicated.

In detecting receptor antagonists or enzyme inhibitors, there are three parameters that often affect the outcome in a predictable quantitative manner: concentrations of the receptors (enzyme), labeled ligand (substrate), and antagonist (inhibitor). The usual goal of assay optimization is to maximize the ability of the assay to detect low concentrations of the analyte. Another question of practical importance, especially in screening of large numbers of samples, would be minimization of the reagent cost. Although the mathematical theory of optimization of the receptor binding assay was developed a long time ago, the resulting formulas (in the general case of unequal affinities of ligand and competitor) were not well suited for practical use. The current availability of computational programs, such as Mathematica, makes possible an efficient solution, both for receptor- and enzyme-based assays. We use a graphical approach to assay optimization and apply it to the following problems: (1) optimization of assay sensitivity, (2) optimization of the reagent cost, and (3) analysis of the entire range of the parameter values since the mathematically optimal values may sometimes be impractical. The computation is extremely simple and the problem can sometimes be solved in several minutes., (Copyright 1999 Academic Press.)

Melatonin binding sites were characterized in mouse peritoneal macrophages. Binding of 2-[125I]melatonin by macrophages fulfills all criteria for binding to a receptor site. Thus, binding was dependent on time, temperature and cell concentration, stable, reversible, saturable and specific. Stoichiometric studies showed a high-affinity binding site with a Kd of 0.58-0.71 nM. These data are in close agreement with data obtained from kinetic studies (Kd = 0.29 nM). The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, binding experiments using macrophage crude membranes showed that melatonin bound specifically to the membranes. Additionally, in competition studies we observed a low-affinity binding site (Kd = 2.02 microM). Melatonin inhibited significantly forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner. This effect was blocked by luzindole, an antagonist of the melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybosilation and Western blot experiments demonstrated both alpha(i1/2) and alpha(i3/o) G protein subunits expression in mouse peritoneal macrophages membranes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal transduction pathway that involves the inhibition of adenylyl cyclase.

To investigate the action of melatonin on the reproductive system, the effect of prolonged versus short-term exposure to melatonin on the release of gonadotrophin releasing hormone (GnRH) was examined in hypothalamic explants of male mink sacrificed in July, September or November. Mediobasal hypothalamic (MBH) explants including the pars tuberalis (PT) were incubated for 1 night with or without melatonin (10(-8) M) for 8 hr or 16 hr and the release of GnRH was then measured. The next day, the explants were incubated further but in a melatonin free buffer, and the release of GnRH was measured with increasing time. Half of the July and September explants had melatonin binding sites quantified by autoradiography. In November, a 16-hr exposure to melatonin induced a significant increase in the release of GnRH during the night, compared with control or 8-hr melatonin exposure. This increase persisted for at least 45 min after the withdrawal of melatonin, suggesting a stimulatory effect of melatonin on the synthesis of GnRH; this effect was apparent in July, September and November. In September, the density of melatonin binding in the PT was significantly lower in the explants incubated for 16 hr with melatonin, compared with those incubated for 8 hr. Thus, in vitro, a long exposure to melatonin, mimicking a single long night, stimulates the release and synthesis of GnRH in parallel with a decrease in the density of melatonin binding in the PT. These effects seem to depend heavily on the duration of exposure to melatonin.

Summer infertility remains a major problem in domestic pigs. It has been proposed that sows which display this trait are inherent seasonal breeders. The influence of photoperiod on domestic pigs has been difficult to ascertain as significant diurnal fluctuations in blood levels of the pineal hormone, melatonin, which provide a direct neuroendocrine transduction of the ambient photoperiod in other species, remain questionable in adult pigs. To investigate whether the pig is potentially receptive to melatonin, central sites of action for this hormone were localized and characterized within the brain and pituitary of the neonatal pig by in vitro autoradiography using 2-((125)I)iodomelatonin. Specific binding was distributed over a number of discrete regions of the brain including the cerebral cortex, hypothalamus, thalamus, brainstem, and cerebellum. The choroid plexus, and the pars tuberalis and pars distalis of the pituitary were also specifically labeled. Specific binding was completely abolished in the presence of 10(-7) M melatonin, and inhibited in the presence of 10(-4) M GTPgammaS (guanosine-5-0-(3-thiotriphosphate)), a non-hydrolysable analogue of GTP, in all regions examined, indicating that binding is representative of a G-protein coupled receptor. Characterization studies showed that 2-((125)I)iodomelatonin binding was saturable with a dissociation constant (Kd) in the low picomolar range (approximately 30 pM). Competition studies with iodomelatonin, melatonin, N-acetylserotonin and serotonin (5-HT) gave IC50 values similar to those previously characterized for the melatonin receptor in the ovine pars tuberalis.

Two 11C-labelled melatonin derivatives, 2-iodo-[11C]melatonin (2-iodo-5-methoxy-N[11C-acetyl]-tryptamine, an agonist) and 2-phenyl-[11C]melatonin (2-phenyl-5-methoxy-N[11C-acetyl]tryptamine, a putative antagonist) were synthesized from [11C]carbon dioxide. The reaction sequence was common to both compounds and consisted of three steps: (i) carbonylation of methyl magnesium bromide with [11C]carbon dioxide, (ii) conversion of the adduct to [11C]acetyl chloride, (iii) acetylation of the amine precursors (2-iodo-5-methoxy-tryptamine or 2-phenyl-5-methoxy-tryptamine) with [11C]acetyl chloride. The precursors were especially prepared. The radiochemical yield was 19% for 2-iodomelatonin and 32% for 2-phenymelatonin, based on [11C]carbon dioxide; the specific activity ranged from 300 to 600 mCi/mumol. Both labelled 2-substituted-melatonins are intended to be used as radiotracers to study melatonin binding sites in man with positron emission tomography.

Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.

Through its secretion of melatonin, the pineal complex of vertebrates exerts a range of physiological effects including regulation of circadian rhythms, seasonal reproduction, metamorphosis, and body color change. Little is known about phylogenetic differences in the distribution and characteristics of melatonin binding sites in fishes. We used in vitro autoradiography to examine binding of [2-125I]iodomelatonin (IMEL) in 20-micron frozen sections of amphioxus (Branchiostoma lanceolatum), Atlantic hagfish (Myxine glutinosa), larval and adult lamprey (Petromyzon marinus), little skate (Raja erinacea), and rainbow trout (Oncorhynchus mykiss). Tissue was incubated with IMEL in the presence or absence of unlabeled melatonin (1 muM, in order to assess nonspecific binding). A concentration of 32 pM IMEL was used for single point assays and competition studies. No specific binding was found in hagfish or amphioxus, which lack a pineal complex. In the optic tecta of lamprey, skate, and trout, IMEL binding is highly specific (melatonin >> N-acetylserotonin > 5- methoxytryptophol >> serotonin). Scatchard analysis revealed that the tectal binding sites are of high affinity (Kd = 36, 38, and 50 pM) and low capacity (Bmax = 8.1, 19.8, and 21.8 fmol/mg protein) in lamprey, skate, and trout, respectively. In adult lampreys, intense specific IMEL binding is found in the optic tectum (layer I > II > III) and preoptic nucleus (pars parvocellularis > magnocellularis). Binding was less intense and consistent in the same areas of ammocoete brain. In skates and trout, intense specific binding is found in optic tectum, lateral geniculate body, diencephalic preoptic and suprachiasmatic nuclei, basal hypothalamus, and the medial pallium. These results indicate that specific melatonin binding sites are present in all craniate taxa examined except in hagfish. Although we cannot rule out the possibility that melatonin receptors are secondarily lost in hagfish, their absence in amphioxus makes this unlikely. We speculate that melatonin actions in early vertebrates may have included regulation of visual and endocrine responses to light., (Copyright 1998 Academic Press.)

Melatonin receptors in the human fetal kidney were identified and characterized by quantitative in vitro autoradiography using the melatonin agonist, 2-[125I]iodomelatonin. Specific binding was localized to cells in the nephrogenic region at the outer perimeter of the developing kidney and was time-dependent, saturable and inhibited in the presence of guanosine 5'-0-(3-thiotriphosphate) indicative of a G protein-coupled receptor. Expression of the Mel1a and Mel1b melatonin receptors in human fetal kidney was determined using RT-PCR. In situ hybridization confirmed the localization of the Mel1a mRNA transcripts. A role for melatonin in development of the human fetal kidney is postulated.

The expression of the melatonin receptor is positively regulated by cAMP and negatively regulated by melatonin in the ovine pars tuberalis (PT). Furthermore, when PT cells are dispersed in primary culture, both messenger RNA (mRNA) and protein levels spontaneously increase through a process that can be blocked by melatonin, but does not involve cAMP. This suggests that other second messengers may be regulated by melatonin, which, in turn, regulates melatonin receptor mRNA and protein levels. In this study using ribonuclease protection assays, ligand binding, protein kinase C (PKC), and cAMP analysis, we demonstrate that the levels of Mel 1a mRNA and protein expression in ovine PT are reduced by phorbol 12-myristate 13-acetate in a cAMP-independent process. This is indicative of an inhibitory role for PKC in receptor regulation. Melatonin, however, does not act through PKC activation to reduce Mel 1a mRNA or protein levels. Basal PKC activity in PT cells can be inhibited by the PKC inhibitor Ro 31-8220, and this suggests that basal PKC activity may suppress Mel 1a receptor expression. Paradoxically, however, Ro 31-8220 also inhibits melatonin receptor mRNA and protein levels in PT cells by a cAMP-independent mechanism. This suggests that other undefined pathways must play an important role in the physiological self-regulation of Mel 1a receptor expression by melatonin.

Red deer (Cervus elaphus) exhibit highly seasonal rhythms in physiology and behaviour. The influence of photoperiod on the timing of these changes begins in utero where the fetus receives photoperiodic information via the diurnal pattern of maternal melatonin secretion. The potential sensitivity of deer fetuses to melatonin was ascertained by mapping specific receptors and characterizing them using 2-[125I]iodomelatonin and quantitative autoradiography in vitro. Specific binding occurred from day 31 of gestation onwards (term = 233 days) over the spinal nerves and respiratory system. At later stages of gestation binding occurred over the brain, particularly the brainstem, the pituitary gland, thyroid gland, gastrointestinal tract including the pancreas, metanephros, cochlea of the ear, spinal cord, and spinal and cranial nerves. Binding was abolished in the presence of 10(-7) mol melatonin l-1 and diminished in the presence of 10(-4) mol GTP gamma S l-1 (guanosine-5-O-(3-thiotriphosphate)), confirming that binding represented functional G-protein-coupled melatonin receptors. Characterization studies, carried out on fetal lung, revealed that binding was time-dependent, reaching equilibrium at about 3 h at room temperature (22 degrees C), and saturable with a dissociation constant (Kd) of 104 pmol l-1. This study demonstrates the presence of G-protein-coupled melatonin receptors over a wide range of tissues in red deer fetuses from early in gestation, indicating that in addition to its role in the communication of photoperiodic information to the fetus in this species, melatonin may be involved in fetal growth and development.

In the present study, we report the localization of the Rev-ErbA alpha and beta nuclear orphan receptors, two closely related members of the nuclear hormone receptor superfamily, in the brain. Both Rev-ErbA variant mRNAs were highly expressed in the olfactory bulb, the hippocampus, and in the granular cells of the cerebellum, areas enriched also in other nuclear orphan receptors. Furthermore, the alpha-isoform was found in high amounts in the frontal cortex, the superficial gray layer of the superior colliculus, and the stria terminalis. Lower expression was observed in the nucleus accumbens, the caudate-putamen, and in some thalamic and brainstem nuclei. The beta-variant, in contrast, was only moderately expressed in the cortex, mainly in the striate and retrosplenial cortices. In addition, moderate levels of Rev-ErbA beta mRNA were seen in various thalamic, pontine and brainstem nuclei. We conclude that the two Rev-ErbA isoforms share a partly similar pattern of expression in the brain, especially in areas that also contain other nuclear orphan receptors and that otherwise the localization of the two receptor subtypes is differential.

To define the hormonal influences that are directly involved in the hair follicle cycles of animals with differing patterns of fibre growth and moulting, we have investigated the possible presence of IGF-I and melatonin receptors on the dermis and hair follicles of cashmere and Angora goats, sampled in February. March and June, using quantitative in vitro autoradiography. The presence of IGF-I receptors in the dermis of both breeds of goat was determined using cryostat sections incubated with 50 pM 125I-labelled IGF-I in the presence or absence of 50 nM IGF-I. Sections of the growing tip of deep antlers concerning the cartilaginous zone, a tissue known to contain high concentrations of specific IGF-I receptors, were used as a positive control. As the production of antler velvet uniquely involves the generation of hair follicles de novo, the presence of IGF-I receptors in the velvet-producing region was also investigated. In both breeds of goat, specific 125I-IGF-I binding was localised over the inner and outer root sheath, the matrix, the germinal matrix, the dermal papilla and the sebaceous glands and satisfied the basic kinetic criteria considered to be representative of a specific IGF-I receptor. Analysis of saturation isotherms using a one-site binding model revealed dissociation constants (Kd) in the range 0.1-0.9 nM and theoretical maximal numbers of binding sites (Bmax) between 21.4 and 45.6 fmol/mg tissue. Kd and Bmax values derived from cashmere and Angora goats sampled at different times of the year did not differ significantly between breeds or sampling times. Specific 125I-IGF-I binding was also localised to the developing follicles on the deer antler dermis. The presence of melatonin receptors within the goat dermis was also investigated. Sections were incubated with 100 pM 2-[125I]iodomelatonin with or without 0.1 microM melatonin, along with sections of sheep pars tuberalis which are known to contain high levels of high-affinity melatonin receptors. No displaceable 2-[125I]iodomelatonin binding was found on any sections of the cashmere or Angora skin analysed. It is therefore concluded that melatonin receptors are not present on the hair follicles or associated structures. IGF-I receptors are present on the hair follicle and sebaceous gland and may be involved in the growth of both seasonally and non-seasonally produced fibre and in the development of antler velvet.

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21-23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

The S. cerevisiae VPS10 (vacuolar protein sorting) gene encodes a type I transmembrane protein of 1577 amino acids required for the sorting of the soluble vacuolar protein carboxypeptidase Y (CPY). Mutations in VPS10 result in the selective missorting and secretion of CPY; all other vacuolar proteins tested are delivered to the vacuole in vps10 mutants. Chemical cross-linking studies demonstrate that Vps10p and the Golgi-modified precursor form of CPY directly interact. A single amino acid change in the CPY vacuolar sorting signal prevents this interaction. Vps10p also interacts with a hybrid protein containing the CPY sorting signal fused to the normally secreted enzyme invertase. Subcellular fractionation indicates that the majority of Vps10p is localized to a late Golgi compartment where vacuolar proteins are sorted. We propose that VPS10 encodes a CPY sorting receptor that executes multiple rounds of sorting by cycling between the late Golgi and a prevacuolar endosome-like compartment.

Low passage (L) T47D cells cultured for up to 12 months in media containing fetal bovine serum, fetal bovine serum plus 1 nM 17 beta-estradiol and estrogen-deficient media gave high passage cells denoted H, H(E+) and H(E-) cells, respectively, which exhibited differential responsiveness to 17 beta-estradiol, various growth factors, tamoxifen, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and their combinations. Moreover, the altered mitogen/antimitogen responsiveness was paralleled by changes in hormone receptor levels, cellular architecture and ploidy. Estrogen receptor binding levels in the H, L and H(E+) cells varied from 36 to 155 fmol/mg protein; in contrast, the estrogen receptor binding in H(E-) cells exhibited a time-dependent increase from 81 to 1229 fmol/mg after culturing in estrogen-deficient media for approximately 12 months. Gel mobility shift assays of the nuclear estrogen receptor extracts from high and low passage cells with 32P-labeled estrogen responsive element showed that levels of the estrogen receptor-estrogen responsive element retarded band were lower in all the high passage cells compared to the low passage cells. These studies further illustrate the genetic instability of the T47D human breast cancer cell line and the resulting changes in mitogen and antimitogen responsiveness. In addition, the high passage H(E-) cells which express high estrogen receptor but low estrogen responsive element binding represent a unique model system for investigating the cellular and molecular biology of breast cancer cells which appear to be estrogen receptor-positive but are insensitive to antiestrogens.