Melatonin Receptor Subtypes Research Articles

Conformation by NMR of two tetralin-based receptor ligands

The conformation in solution of 1-phenyl-3-propionamido-1,2,3,4-tetrahydronaphthalene and 1-phenyl-3-( N, N-dimethylamino)-1,2,3,4-tetrahydronaphthalene has been determined by a combination of nuclear magnetic resonance measurements and molecular mechanics calculations. The results indicate that in the cis isomers the cyclohexene ring is in a locked conformation and the trans isomers correspond to a mixture of the two inverted half chairs. Moreover, the data allowed the identification of the two purposely-synthesized geometrical isomers of 1-phenyl-3-propionamidotetralin. Binding studies on melatonin receptor subtypes showed that the (±)- cis-1-phenyl-3-propionamido-1,2,3,4-tetrahydronaphthalene has higher affinity and selectivity ratio toward the MT 2 subtype than the (±)- trans-isomer.

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Our previous work showed that melatonin (N-acetyl-5-methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[125I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[125I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.

Melatonin binding sites in estrogen receptor‐positive cells derived from human endometrial cancer

Abstract: Our previous work showed that melatonin (N‐acetyl‐5‐methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor‐positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2‐[125I]‐iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2‐[125I]‐iodomelatonin at all concentrations tested (10−8 to 10−4 m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.

Melatonin receptors and their regulation: biochemical and structural mechanisms

There is growing evidence demonstrating the complexity of melatonin's role in modulating a diverse number of physiological processes. This complexity could be attributed to the fact that melatonin receptors belong to two distinct classes of proteins, that is, the G-protein coupled receptor superfamily (MT 1, MT 2) and the quinone reductase enzyme family ( MT 3 ) which makes them unique at the molecular level. Also, within the G-protein coupled receptor family of proteins, the MT 1 and MT 2 receptors can couple to multiple and distinct signal transduction cascades whose activation can lead to unique cellular responses. Also, throughout the 24-hour cycle, the receptors' sensitivity to specific cues fluctuates and this sensitivity can be modulated in a homologous fashion, that is, by melatonin itself, and in a heterologous manner, that is, by other cues including the photoperiod or estrogen. This sensitivity of response may reflect changes in melatonin receptor density that also occurs throughout the 24-hour light/dark cycle but out of phase with circulating melatonin levels. The mechanisms that underlie the changes in melatonin receptor density and function are still not well-understood, but data is beginning to show that transcriptional events and G-protein uncoupling may be involved. Even though this area of research is still in its infancy, great strides are being made everyday in elucidating the mechanisms that underlie melatonin receptor function and regulation. The focus of this review is to highlight some of these discoveries in an attempt to reveal the uniqueness of the melatonin receptor family while at the same time provide thought-provoking ideas to further advance this area of research. Thus, a brief overview of each of the mammalian melatonin receptor subtypes and the signal transduction cascades to which they couple will be discussed with a greater emphasis placed on the mechanisms underlying their regulation and the domains within the receptors essential for proper signaling.

Targeted disruption of the mouse Mel(1b) melatonin receptor.

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

Differential coupling of the extreme C-terminus of G protein α subunits to the G protein-coupled melatonin receptors

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Gα subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gαs chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Gα subunits. The Gαs portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT 1 or MT 2 melatonin receptors with Gαs chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Gα C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Gα chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gαs chimaeras coupled. Recognition of the C-terminal five amino acids of the Gα subunit is a requisite for coupling to a receptor, but it is not the sole determinant.

Differential expression of high-affinity melatonin receptors (MT1) in normal and malignant human breast tissue.

Melatonin is a pineal hormone that strongly inhibits the growth of breast cancer cells in vitro and in vivo. We report thefirst use of immunohistochemical analysis to determine the distribution of the high-affinity melatonin receptor subtype, MTI, in human breast tissue, the hypothalamic suprachiasmatic nucleus, and skin. The MT1 antibody, which is specific for the cytoplasmic portion of the receptor, produced cytoplasmic staining in normal-appearing breast epithelial cells and ductal carcinoma cells; stromal cells, myoepithelial cells, and adipocytes were nonreactive. The majority of nonneoplastic samples (13/19 [68%]) were negative to weakly positive, while moderate to strong reactivity was seen in most cancer samples (49/65 [75%]). Thus, although MT1 receptors were detectable in normal and malignant breast epithelium, high receptor levels occurred more frequently in tumor cells (P < .001), and tumors with moderate or strong reactivity were more likely to be high nuclear grade (P < .045). These findings may have implications for the use of melatonin in breast cancer therapy.

Mammalian melatonin receptors: molecular biology and signal transduction.

The pineal hormone, melatonin, is an important regulator of seasonal reproduction and circadian rhythms. Its effects are mediated via high-affinity melatonin receptors, located on cells of the pituitary pars tuberalis (PT) and suprachiasmatic nucleus (SCN), respectively. Two subtypes of mammalian melatonin receptors have been cloned and characterized, the MT1 (Mel(1a)) and the MT2 (Mel(1b)) melatonin receptor subtypes. Both subtypes are members of the seven-transmembrane G protein-coupled receptor family. By using recombinant melatonin receptors it has been shown that the MT1 melatonin receptor is coupled to different G proteins that mediate adenylyl cyclase inhibition and phospholipase C beta activation. The MT2 receptor is also coupled to inhibition of adenylyl cyclase and additionally it inhibits the soluble guanylyl cyclase pathway. In mice with a targeted deletion of the MT1 receptor, the acute inhibitory effects of melatonin on SCN multiunit activity are completely abolished, while the phase-shifting responses to melatonin (given in physiological concentrations) appear normal. Furthermore, melatonin inhibits the phosphorylation of the transcription factor cyclic AMP response element binding protein, induced by the pituitary adenylate cyclase-activating polypeptide in SCN cells predominantly via the MT1 receptor. However, a functional MT2 receptor in the rodent SCN is partially able to compensate for the absence of the MT1 receptor in MT1 receptor-deficient mice. These findings indicate redundant and non-redundant roles of the receptor subtypes in regulating SCN function. In the PT, a functional MT1 receptor is essential for the rhythmic synthesis of the clock gene product mPER1. Melatonin produces a long-lasting sensitization of adenylyl cyclase and thus amplifies cyclic AMP signaling when melatonin levels decline at dawn. This action of melatonin amplifies gene expression rhythms in the PT and provides a mechanism for reinforcing rhythmicity in peripheral tissues which themselves lack the capacity for self-sustained oscillation. Mice with targeted deletion of melatonin receptor subtypes provide an excellent model to understand cellular mechanisms through which melatonin modulates circadian and photoperiodic rhythmicity.

Synthesis and structure-affinity-activity relationships of novel benzofuran derivatives as MT(2) melatonin receptor selective ligands.

A series of N-(2-phenylbenzofuran-3-yl) ethyl amide and N-(2-arylalkylbenzofuran-3-yl) ethyl amide derivatives were synthesized and evaluated as melatonin receptor ligands. The affinity of each compound for the two MT(1) and MT(2) melatonin receptor subtypes was determined by binding studies using 2-[(125)I]iodomelatonin on human embryonic kidney cell line HEK293 membrane homogenates. The intrinsic activity of the most interesting compounds was evaluated on the [(35)S]GTPgammaS binding assay. Introduction of a 2-phenyl substituent in the C-2 benzofuran position leads to an agonist compound, 10q, which binds more strongly than melatonin itself to both MT(1) and MT(2) subtypes. On the other hand, a 2-benzyl group in the same position allows MT(2) antagonist selective ligands to be obtained. The MT(2) selectivity and antagonist potency can be modulated with suitable modifications on the N-acyl and benzyl substituents, and the most selective compounds 10c and 19 show affinity ratios of 123 and 192, respectively, and bind to the MT(2) subtype similarly to melatonin itself (0.1 nM). Nevertheless, 10c acts as an MT(1) and MT(2) antagonist, whereas 19 is a partial agonist.

MT1 melatonin receptor mRNA expression exhibits a circadian variation in the rat suprachiasmatic nuclei

The aim of the present study was to investigate the daily regulation of both MT1 and MT2 melatonin receptor subtype mRNA expression in the rat SCN in order to clarify their role in the daily variation of SCN melatonin receptors. Existing MT1 and MT2 partial clones were extended by PCR to 982 and 522 bp, respectively. However, while the MT1 clone allowed us to set up a highly sensitive in situ hybridization (ISH) method, we could not detect MT2 expression within the SCN. Therefore, our results suggest that only MT1 mRNA can be correlated with 2-iodo-melatonin binding sites in the rat SCN. Investigation of MT1 mRNA expression throughout the 24 h light/dark cycle or in constant darkness clearly showed that in the two conditions, mRNA expression showed a robust rhythm with two peaks, one after the day/night and one after the night/day transitions in LD, and at the beginning of the subjective night and day in DD, respectively. Furthermore, these variations were not linked to the daily changes in melatonin receptor density. Thus, the transcriptional regulation of MT1 receptors does not appear to play a role in the daily regulation of melatonin binding sites availability.

Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells.

In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05-1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinson's disease.

Melatonin Receptor mRNA and Protein Expression in Xenopus laevis Nonpigmented Ciliary Epithelial Cells

Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel1c melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel1c but not Mel1b receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.

Direct action of melatonin in human granulosa-luteal cells.

The direct involvement of melatonin in modulation of ovarian steroidogenesis, the high levels of melatonin found in human follicular fluid, and the presence of melatonin binding sites in the ovary led us to hypothesize that melatonin acts as a modulator of ovarian function. In contrast to the hypothalamus and pituitary, the mechanism of melatonin action at the level of the ovary is still poorly understood. In the present study, we investigated the gene expression of the two different forms of melatonin receptors in human granulosa-luteal cells, using RT-PCR. PCR products corresponding to the expected sizes of the melatonin receptor subtypes, mt(1)-R and MT(2)-R, were obtained from granulosa-luteal cells, and the authenticity of the PCR products was confirmed by Southern blot hybridization with cDNA probes. Subsequent cloning and sequence analysis revealed that the ovarian mt(1)-R and MT(2)-R cDNAs are identical to their brain counterparts. Because gonadotropins and GnRH acting through specific receptors in the human ovary regulate cellular functions, we investigated the role of melatonin in the regulation of FSH receptor, LH receptor, GnRH, and GnRH receptor levels. Treatment with melatonin (10 pM-100 nM) significantly increased LH receptor mRNA levels without altering the expression of the FSH receptor gene. Both GnRH and GnRH receptor mRNA levels were significantly decreased, to 61% and 45% of control levels, respectively, after melatonin treatment. Melatonin treatment alone had no effect on basal progesterone production but enhanced the effects of human CG-stimulated progesterone production. Because MAPKs are activated in response to a diverse array of extracellular stimuli leading to the regulation of cell growth, division, and differentiation, and because melatonin has been shown to modulate cellular proliferation and differentiation, in this study, we demonstrated that melatonin activated MAPK in a dose- and time-dependent manner. In summary, our studies demonstrate, for the first time, that melatonin can regulate progesterone production, LH receptor, GnRH, and GnRH receptor gene expression through melatonin receptors in human granulosa-luteal cells, which may be mediated via the MAPK pathway and activation of Elk-1. Our results support the notion that melatonin plays a direct role in regulating ovarian function.

Melatonin receptor RNA is expressed in photoreceptors and displays a diurnal rhythm in Xenopus retina

Melatonin is an output signal of an endogenous circadian clock of retinal photoreceptors, with highest levels occurring at night. Melatonin synthesized in the retina appears to act as a paracrine signal by binding to specific receptors in the eye. We have previously demonstrated that RNA encoding the Mel 1b and Mel 1c melatonin receptor subtypes is expressed in the Xenopus laevis retina. The goal of this study was to determine the distribution of the Mel 1b and Mel 1c receptor subtype RNA expression in the retina, and to determine if the level of expression of these receptors exhibits a diurnal rhythm. Sections of frog neural retina were analyzed by in situ hybridization with 35S-labeled Xenopus Mel 1c and Mel 1b riboprobes. Hybridization was present in cells of the inner nuclear layer and the ganglion cell layer. Moreover, there was hybridization in the photoreceptors, which has not been previously reported. To test the hypothesis that retinal melatonin receptor mRNA undergoes a diurnal rhythm of expression, total RNA was isolated from frog neural retinas obtained at 3-h intervals during a 24-h period. The total RNA was used in real-time PCR assays to quantify the differences in Mel 1b and Mel 1c receptor mRNA expression at various circadian times. Both the Mel 1b and Mel 1c receptor RNA demonstrated a diurnal rhythm of expression, with peak levels occurring late in the light period, and lowest levels late in the dark period. These results support the hypothesis that RNA encoding melatonin receptors undergo a diurnal rhythm of expression. To further investigate the possible expression of the Mel 1a receptor subtype in Xenopus retina, we generated Mel 1a PCR products in genomic DNA, and in reverse-transcribed neural retina and retinal pigment epithelium (RPE) RNA. The identity of the PCR product was confirmed by sequencing. Therefore, all three known Xenopus melatonin receptor subtypes appear to be expressed in the neural retina and RPE.

Characterization of 2-[125I]-Iodomelatonin Binding Sites in the Golden Hamster Retina by Autoradiography

The characterization of 2-[125I]-iodomelatonin binding sites was performed in the golden hamster retina using in vitro quantitative autoradiography. The specific binding of the radioligand fulfills all the criteria for binding to a receptor site, being stable, reversible, saturable and of high affinity. 2-[125I]-iodomelatonin labels a single class of sites in the sections of whole eyes as well as in isolated retinas with a similar affinity, whereas the total number of receptors was higher in sections of whole eyes than in isolated retinas. Melatonin and related analogues competed for 2-[125I]-iodomelatonin binding with the following order of affinities: 2-iodomelatonin > 6-hydroxymelatonin > melatonin > 6-chloromelatonin >>> N-acetyl-5-hydroxytryptamine (NAS) > 5-methoxytryptamine > 5-hydroxytryptamine (serotonin). Micro-molar concentrations of GTP and GDP dose-dependently and specifically inhibited agonist binding, suggesting coupling of the binding sites to a Gi protein. These results suggest the participation of melatonin in the regulation of retinal physiology trough activation of melatonin receptor subtypes.

Galpha(14) links a variety of G(i)- and G(s)-coupled receptors to the stimulation of phospholipase C.

1. The bovine Galpha(14) is a member of the G(q) subfamily of G proteins that can regulate phospholipase Cbeta isoforms but the extent to which Galpha(14) recognizes different receptor classes is not known. 2. Galpha(14) was cotransfected with a variety of receptors in COS-7 cells, and agonist-induced stimulation of phospholipase C was then measured. 3. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Galpha(14) stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. 4. Among the three opioid receptors (mu, delta, and kappa), only the delta receptor was capable of stimulating IP formation when coexpressed with Galpha(14) in COS-7 cells. 5. A panel of G(i)- and G(s)-linked receptors was screened for their ability to stimulate IP accumulation via Galpha(14). The adenosine A(1), complement C5a, dopamine D(1), D(2) and D(5), formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Galpha(14), while the alpha(2)- and beta(2)-adrenoceptors were able to do so. 6. Galpha(14)-mediated stimulation of phospholipase Cbeta was agonist dose-dependent. These data demonstrate that although Galpha(14) can interact with different classes of receptors, it is much less promiscuous than Galpha(15) or Galpha(16).

24h VARIATION IN THE EXPRESSION OF THE mt1 MELATONIN RECEPTOR SUBTYPE IN CORONARY ARTERIES DERIVED FROM PATIENTS WITH CORONARY HEART DISEASE

Previous studies presented evidence for impaired nocturnal secretion and synthesis of melatonin in patients with coronary heart disease (CHD). This study aimed to investigate whether the melatonin receptor subtype mt1 is differentially expressed in coronary arteries derived from patients with CHD (n = 9) compared to patients with dilative cardiomyopathy (CMP; n = 10) who served as controls. Expression of the mt1 receptor was studied in sections of isolated coronary arteries by a reverse transcriptase–polymerase chain reaction (RT-PCR) and a Western immunoblot technique. In addition, the data from the Western blotting of 15 patients were interpolated against the exact time of aortic clamp to study the 24h expression of the mt1 receptor. The analyses of the results from both methods indicated the presence of the mt1 receptor in all of the individuals. No statistically significant difference was observed in the receptor expression between patients with CHD and those with CMP (in arbitrary units: 3.39 ± 3.08 versus 3.91 ± 2.78). Expression of the melatonin receptor in the coronary arteries of the whole patient group presented a 24h variation, with the lowest values detectable after 02:00 up to the late morning hours and a progressive increase beginning after 13:00 until 00:00 (mesor = 3.66, amplitude = 3.23, acrophase = 20.45, P =. 0003). When studying the 24h variation in patients with CHD and CMP separately, a nearly similar circadian course was observed. In conclusion, we demonstrated for the first time a 24h variation of a melatonin receptor subtype in human vessels. Furthermore, in relation to our results, we suggest that the expression of the mt1 melatonin receptor in the coronary arteries is probably not impaired in patients with CHD. (Chronobiology International, 18(6), 973–985, 2001)

Characterization of an Antibody to the Human Melatonin mt1 Receptor

AbstractMelatonin 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 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.

Modulation of Blood Glucose by Melatonin: A Direct Action on Melatonin Receptors in Mouse Hepatocytes

Melatonin receptors were studied in isolated mouse hepatocytes using the 2[¹²⁵I]iodomelatonin binding assay. The binding of 2[¹²⁵I]iodomelatonin to hepatocytes isolated from the mouse using collagenase was stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (K_d) obtained from saturation studies was 10.0 ± 0.4 pmol/l (n = 16), which was comparable to the K_d obtained from kinetics studies (6.9 ± 1.2 pmol/l, n = 3), and the maximum number of binding sites (B_max) was 2.9 ± 0.4 fmol/mg protein (n = 16). The relative order of potency of indoles in competing for 2[¹²⁵I]iodomelatonin binding was 2-iodomelatonin > 2-phenylmelatonin > 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that the binding was mediated by the ML₁ receptor subtype. The linear Rosenthal plots, the close proximity of the Hill coefficient to unity and the monophasic competition curves suggest that a single class of 2[¹²⁵I]iodomelatonin binding sites is present in the mouse hepatocytes. Guanosine 5′-O-(3-thiotriphosphate) dose-dependently inhibited 2[¹²⁵I]iodomelatonin by lowering the affinity of binding, while no inhibitory effects of adenosine nucleotides were observed, suggesting that the binding sites are G-protein linked. Western immunoblotting was used to identify the melatonin receptor subtype in mouse hepatocytes using anti-Mel_1a and anti-Mel_1b. Hepatocyte membrane extract reacted with anti-Mel_1b but not anti-Mel_1a giving a peptide-blockable band of 36 kD, supporting the hypothesis that the melatonin receptors in mouse hepatocytes are of the Mel_1b subtype. Melatonin injection and a high plasma glucose level affected 2[¹²⁵I]iodomelatonin binding in the whole mouse liver homogenates. Plasma glucose was elevated by mid-light intraperitoneal injection of melatonin (4 and 40 mg/kg body weight) in a dose-dependent manner with maximum elevation achieved 1 h after injection. 2[¹²⁵I]Iodomelatonin binding at this time showed increased K_d with no changes in B_max. When the plasma glucose returned to normal within 2 h, the binding remained lowered with increased K_d but no changes in B_max. Elevation of plasma glucose by 2-deoxyglucose injection (500 mg/kg), on the other hand, decreased the binding by decreasing the B_max without affecting the K_d. Suppression of plasma glucose by insulin injection (3 IU/kg) did not change the binding. Thus, melatonin may act directly on the liver to elevate the plasma glucose level, and changes in plasma glucose level itself may in turn affect hepatic melatonin binding.

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, 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.