Janice E. Drew

Localization of the Melatonin-Related Receptor in the Rodent Brain and Peripheral Tissues

Previous studies have provided a limited examination of the expression of the orphan melatonin‐related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin‐related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase‐polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin‐related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

Central melatonin receptors in the rainbow trout: Comparative distribution of ligand binding and gene expression †

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 Mel1a receptor subtype, and one corresponded to the Mel1b 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 hypothalamopituitary axis is not clearly indicated. J. Comp. Neurol. 409:313–324, 1999.

Melatonin receptors in human fetal brain: 2-[125I]iodomelatonin binding and MT1 gene expression

Abstract: The purpose of this study was to identify sites of action of melatonin in the human fetal brain by in vitro autoradiography and in situ hybridization. Specific, guanosine triphosphate (GTP) sensitive, binding of 2‐[125I]iodomelatonin was localized to the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalalmus, specific binding was present in the suprachiasmatic nuclei (SCN) as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei. The localization of MT1 receptor subtype gene expression as determined by in situ hybridization matched the localization of 2‐[125I]iodomelatonin binding. No MT2 receptor subtype gene expression was detected using this technique. Thus, melatonin may act on the human fetus via the MT1 receptor subtype at a number of discrete brain sites. A major site of action of melatonin in both fetal and adult mammals is the pars tuberalis of the pituitary gland. However, no 2‐[125I]iodomelatonin binding or melatonin receptor gene expression was detected in the pituitary gland in the present study, indicating that the pituitary, particularly the pars tuberalis, is not a site of action of melatonin in the human fetus.

Anti-Inflammatory Implications of the Microbial Transformation of Dietary Phenolic Compounds

Due to the success of therapeutic anti-inflammatory compounds to inhibit, retard, and reverse the development of colon cancer, the identification of dietary compounds as chemopreventives is being vigorously pursued. However, an important factor often overlooked is the metabolic transformation of the food-derived compounds in the gut that may affect their bioactivity. Commonly consumed dietary phenolics (esterified ferulic acid and its 5-5′-linked dimer), which have the potential to undergo predominant microbial transformations (de-esterification, hydrogenation, demethylation, dehydroxylation, and dimer cleavage), were incubated with human microbiota. The metabolites were identified (high-performance liquid chromatography and nuclear magnetic resonance) and confirmed to be present in fresh fecal samples from 4 human volunteers. The potential anti-inflammatory properties were compared by measuring the ability of the parent compounds and their metabolites to modulate prostanoid production in a cell line in which the inflammatory pathways were stimulated following a cytokine-induced insult. The compounds were readily de-esterified and hydrogenated, but no dimer cleavage occurred. Only the monomer underwent demethylation and selective de-hydroxylation. The resultant metabolites had differing effects on prostanoid production ranging from a slight increase to a significant reduction in magnitude. This suggests that the microbial transformation of dietary compounds will have important inflammatory implications in the chemoprevention of colon cancer.

The Ovine Melatonin-Related Receptor: Cloning and Preliminary Distribution and Binding Studies

A melatonin‐related receptor was cloned from an ovine genomic library. The sequenced gene has a similar structure to that of the melatonin receptor gene family and consists of two exons separated by an intron of approximately 3u2003kb. Exon 1 and exon 2 of the ovine melatonin‐related receptor encode a protein of 575 amino acids which is 73.8% homologous to the human melatonin‐related receptor and shows 40.9% homology with the ovine Mel1a melatonin receptor. COS‐7 cells transiently expressing ovine melatonin‐related receptors did not bind 2‐[125I]iodomelatonin or 3H‐melatonin. Reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization studies revealed expression of the ovine melatonin‐related receptor in the hypothalamus, pituitary, retina and retinal pigment epithelium. Furthermore, expression of the ovine melatonin‐related receptor is shown to be coincident with Mel1a and 2‐[125I]iodomelatonin binding in the pituitary and serotonin N‐acetyl transferase (arylalkylamine N‐acetyl transferase, AANAT) expression in the retina. Expression patterns and similarity with the melatonin receptor gene family suggest a role for this novel G protein‐coupled receptor in control and regulation of endocrine function and retinal physiology.

Characterisation of human melatonin mt1 and MT2 receptors by CRE-luciferase reporter assay

A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.

Chimeric Melatonin mt1 and Melatonin-Related Receptors: Identification of Domains and Residues Participating in Ligand Binding and Receptor Activation of the Melatonin mt1 Receptor

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt1 receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[125I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[125I]iodomelatonin binding. The subsequent testing of a “reverse” chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt1receptor sequences identified specific 2-[125I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt1 receptor. This identified Gly258 (Gly6.55) as a critical residue required for high affinity ligand binding and receptor function.

Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein‐coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels

Binding assays using 2‐[125I]iodomelatonin revealed high‐affinity, guanosine 5′‐O‐(3‐thiotriphosphate) sensitive, melatonin binding sites (B max 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT‐PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 μM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over‐express the human Mel1a melatonin receptor (B max>400 fmol/mg protein) melatonin dose‐dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein‐coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.

Characterization of an antibody to the human melatonin mt1 receptor.

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, mt1338−352). 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‐(125I)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 63u2003kDa and one at 86u2003kDa. 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.

Identification and characterisation of 2-[125I]iodomelatonin binding and Mel1a melatonin receptor expression in the human fetal leptomeninges

Melatonin binding sites were identified over the leptomeninges surrounding the human fetal brain using quantitative in vitro autoradiography and the melatonin agonist, 2-[125I]iodomelatonin. Binding was found to be saturable and of high affinity (dissociation constant (Kd) = 54 pM and maximal theoretical binding (Bmax) = 13 fmol/mg protein), and inhibited by guanosine-5-o-(3-thiotriphosphate) (GTPgammaS) suggesting that these binding sites represent G protein-coupled melatonin receptors. RT-PCR performed on mRNA isolated from the human fetal leptomeninges detected expression of the G protein-coupled melatonin receptor Mel1a, but not Mel1b. In situ hybridisation confirmed the localisation of Mel1a mRNA transcripts over the leptomeninges of the fetal brain. The identification of 2-[125I]iodomelatonin and Mel1a melatonin receptor expression in the fetal leptomeninges implies that melatonin may play a role in the early growth and development of the human brain.