Molecular Cloning and Pharmacological Characterization of … · 2008. 12. 31. · respectively. Saturation binding experiments with 2-[125I] iodomelatonin revealed that the dissociation

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Molecular Cloning and Pharmacological Characterization of Monkey

MT1 and MT2 Melatonin Receptors Showing High Affinity for the

Agonist Ramelteon

Keiji Nishiyama, Yasushi Shintani, Keisuke Hirai, Shin-ichi Yoshikubo

Pharmacology Research Laboratory, Pharmaceutical Research Division,

Takeda Pharmaceutical Company Ltd., Osaka 532-8686, Japan

(K.N., Y.S., K.H., S.Y.)

JPET Fast Forward. Published on June 25, 2009 as DOI:10.1124/jpet.109.155283

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 25, 2009 as DOI: 10.1124/jpet.109.155283

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Running title: Ramelteon is a potent melatonin receptor agonist in monkeys.

Corresponding Editor: Shin-ichi Yoshikubo, Ph.D., Pharmacology Research

Laboratory, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd,

17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan

Phone: +81-6-6300-6647, Fax: +81-6-6300-6306, E-mail:

[email protected]

Number of pages of text: 35

Tables: 2

Figures: 8

Number of references: 40

Number of words in abstract: 217

Words in introduction: 683

Words in discussion: 963

Abbreviations: PCR, polymerase chain reaction; cAMP, adenosine 3′,5′-cyclic

monophosphate; CHO, Chinese hamster ovary; SDS-PAGE, sodium dodecyl

sulfate-polyacrylamide gel electrophoresis; PACAP, pituitary adenylate

cyclase-activating polypeptide; DMSO, dimethyl sulfoxide; Ramelteon (TAK-375),

(S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl] propionamide

A recommended section assignment: Neuropharmacology


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Abstract

Melatonin receptor agonists such as melatonin and ramelteon (TAK-375) have

sleep-promoting effects in humans. In preclinical models, these effects are more

similar to those observed in monkeys than in other species. However, in contrast to

the human melatonin receptors, the pharmacological characteristics of the monkey

melatonin receptors have yet to be elucidated. In this study, we cloned the

cynomolgus monkey MT1 and MT2 melatonin receptors based on rhesus monkey

genome sequences and then characterized the monkey melatonin receptors and

compared their pharmacological properties with those of the human homologues. The

overall amino acid sequences of the monkey MT1 and MT2 melatonin receptors

showed high homology to the human MT1 (95%) and MT2 (96%) receptors,

respectively. Saturation binding experiments with 2-[125I] iodomelatonin revealed that

the dissociation constant (Kd) for the monkey MT1 and MT2 melatonin receptors were

19.9 and 70.4 pM, respectively. In ligand competition assays using 2-[125I]

iodomelatonin, ramelteon displayed approximately 3- to 7-fold higher affinities than

melatonin for the recombinant monkey MT1, and MT2 melatonin receptors and

monkey suprachiasmatic nucleus membranes. This higher affinity of ramelteon

compared to melatonin has also been observed in human melatonin receptors.

Furthermore, ramelteon inhibited PACAP27-stimulated cAMP production with higher

potency than melatonin. In conclusion, this information will help us to understand the

pharmacological effects of melatonin receptor agonists in monkeys.


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Introduction

The hormone melatonin is produced primarily in the mammalian pineal gland and is

released in a circadian manner, with low levels during the day and high levels at night

(Tamarkin et al., 1985; Foulkes et al., 1997). Melatonin has been implicated in numerous

physiological processes, including the sleep-wake cycle, circadian entrainment, and

seasonal reproduction (Reiter, 1980; Redman et al., 1983; Bartness et al., 1993; Slotten et

al., 1999; Wyatt et al., 2006). These effects are achieved by the binding of melatonin to

specific receptors in the suprachiasmatic nucleus (SCN) of the hypothalamus (Zee and

Manthena, 2007) and in the pars tuberalis (PT) of the pituitary (Morgan, 2000).

Two high affinity melatonin receptor types, MT1 and MT2, have been cloned in

mammals: human, sheep, Siberian hamster, mouse, and rat (Reppert et al., 1994, 1995;

Weaver et al., 1996; Roca et al., 1996; Jin et al., 2003; Audinot et al., 2008). The MT1 and

MT2 melatonin receptors exhibit different molecular structure and chromosomal

localization among these species. The human MT1 and MT2 melatonin receptors exhibit

only 60% overall identity at the amino acid level, and show distinct binding profiles; the

human MT2 melatonin receptor has a lower affinity for 2-[125I] iodomelatonin compared to

the human MT1 melatonin receptor (Reppert et al., 1995; Dubocovich et al., 1997; Audinot

et al., 2003). Both receptors belong to the family of G-protein-coupled receptors that are

associated with the inhibition of adenylate cyclase (Reppert et al., 1994, 1995). Furthermore,

activation of the MT1 melatonin receptor induces a transient elevation in cytosolic calcium

ions and inositol phosphate concentration, whereas activation of the MT2 melatonin

receptor inhibits the soluble guanylate cyclase pathway in MT2 receptor-transfected cells

(Brydon et al., 1999; Petit et al., 1999; MacKenzie et al., 2002).

The expression of melatonin receptors has been studied in various tissues using

autoradiography with 2-[125I] iodomelatonin, in situ hybridization, and reverse


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transcription-polymerase chain reaction (RT-PCR). The MT1 melatonin receptor is

expressed most abundantly in the SCN, and is also expressed in other regions of the brain

(e.g., the paraventricular thalamus) and some peripheral tissues (e.g., the pituitary, kidney,

and small intestine) (Reppert et al., 1994; Roca et al., 1996; Drew et al., 1998; Sallinen et

al., 2005). A targeted deletion of the MT1 melatonin receptor in mice has been demonstrated

to block the melatonin-induced inhibition of neuronal firing in SCN slices; however, the

phase-shifting responses to melatonin were only slightly impaired (Liu et al., 1997). The

MT2 receptor is expressed dominantly in the retina, and also in the SCN and hippocampus

(Reppert et al., 1995; Liu et al., 1997; Dubocovich et al., 1998). Activation of the MT2

melatonin receptor phase-shifts the circadian rhythm of neuronal activity in the SCN and

inhibits dopamine release in the retina (Dubocovich et al., 1997, 1998, 2005).

Accumulating evidence suggests that melatonin can influence sleep-promoting and

sleep-wake rhythm regulating action through MT1 and MT2 melatonin receptors. Although

studies of the sleep-promoting effect of melatonin have not always yielded consistent

results, melatonin and ramelteon have been shown to have sleep-promoting effects in cats

(Miyamoto et al., 2004), monkeys (Zhdanova et al., 2002; Yukuhiro et al., 2004), and

humans (Wyatt et al., 2006; Zammit et al., 2007). Indeed, ramelteon has been approved in

the US for the treatment of insomnia. In preclinical studies, monkeys may be the most

suitable species for evaluating the sleep-promoting effect of melatonin receptor agonists.

This is because such an effect has been disputed in nocturnal animals, such as rats (Mailliet

et al., 2001; Akanmu et al., 2004). Decreases in sleep latency after the administration of

melatonin receptor agonists has been demonstrated only in monkeys and humans. However,

the pharmacological profiles of the monkey melatonin receptors remain to be determined.

Therefore, characterization of the monkey melatonin receptors is important for evaluation

and interpretation of the sleep-promoting effect in monkeys. In this study, we cloned the


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MT1 and MT2 melatonin receptors of the cynomolgus monkey and analyzed their

pharmacological characteristics using melatonin and ramelteon. The results of this study

indicate that the pharmacological profiles of the monkey melatonin receptors are

comparable to those of the human melatonin receptors.


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Methods

Chemicals and Drugs

Ramelteon, (S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]

propionamide (TAK-375), was synthesized at Takeda Pharmaceutical Company, Ltd.

(Osaka, Japan) (Uchikawa et al., 2002). Melatonin was purchased from Sigma-Aldrich (St.

Louis, MO) and 2-iodomelatonin was obtained from Tocris Cookson Ltd. (Bristol, UK).

2-[125I] iodomelatonin was obtained from Perkin-Elmer Inc. (Wellesley, MA).

Animals

Twelve adult (9 males and 3 females) cynomolgus monkeys (Macaca fascicularis) that

weighed between 2.8 and 6.6 kg were cared for in accordance with the principles and

guidelines of the Takeda experimental animal committee.

Genomic DNA and RNA isolation

The monkeys were anesthetized with ketamine and then sacrificed. Brain tissues (SCN,

cerebral cortex, putamen, caudate nucleus, amygdala) and eyes were dissected immediately

after obtaining coronal sections of the brains, frozen quickly in dry ice, and then stored at

–80°C until use. Genomic DNA was isolated from brain tissue (putamen) using a QIAGEN

Genomic-tip 100/G and a QIAGEN Proteinase K (QIAGEN, Valencia, CA) according to the

manufacturer’s protocol. Total RNA was extracted from brain tissues and eyes using Isogen

(Nippon Gene, Tokyo, Japan), and then mRNA was isolated using a μMACS mRNA

Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol.

Cloning of monkey MT1 melatonin receptor cDNA

The coding region of monkey MT1 melatonin receptor, which comprises two exons (exon

1 and exon 2), was obtained by nested PCR amplification of monkey genomic DNA using

primers that anneal outside the coding region of each exon. The primer design was based on

an alignment of the sequences of human MT1 melatonin receptor cDNA and the rhesus


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monkey (Macaca mulatta) genome obtained from the University of California at Santa Cruz

(UCSC) genome browser (http://www.genome.ucsc.edu/). Using MT1-exon 1-1F/1R

primers (Table 1), the first PCR of exon 1 was performed in a total volume of 25 µl

containing 0.5 µl of AccuPrime GC-Rich DNA Polymerase (Invitrogen, Carlsbad, CA), 5 µl

of 5× buffer A (300 mM Tris-HCl, pH 9.2, 10 mM MgSO4, 150 mM NaCl, 1 mM dNTPs),

0.5 µl of 10 µM each primer, and 2 µl of genomic DNA (88 ng) under the conditions

described in Table 1. The second PCR was then performed under the same conditions as the

first PCR using 2 µl of a 1:400 dilution of the first-round PCR products as a template and

MT1-exon 1-2F/2R primers (Table 1). Using MT1-exon 2-1F/1R or MT1-exon 2-2F/2R

primers, the first and second PCR amplification was carried out in a total volume of 25 µl

containing 0.5 µl of Pfx50 DNA Polymerase (Invitrogen), 2.5 µl of 10× Pfx50 PCR mix,

0.75 µl of 10 mM dNTPs, 2 µl of 3.75 µM each primer, 2 µl of genomic DNA (88 ng) for

the first PCR or 1:400 dilution of the first-round PCR products for the second PCR under

the conditions described in Table 1. The full-length monkey MT1 melatonin receptor cDNA

was generated by PCR using exon 1 and exon 2 fragments with each other’s partial exon

sequence. Using MT1-exon 1-F3/R3 or MT1-exon 2-F3/R3 primers (Table 1), each

appended exon fragment was obtained by PCR under the conditions described in Table 1. In

order to conjugate the two exon fragments, PCR amplification was carried out using

MT1-exon 1-F3 and MT1-exon 2-R3 primers (Table 1) under the conditions described in

Table 1.

Cloning of monkey MT2 melatonin receptor cDNA

In order to amplify monkey MT2 melatonin receptor cDNA from monkey eye, 139 ng of

monkey eye mRNA was primed with oligo (dT)20 primer and reverse-transcribed according

to the manufacturer’s instructions (Superscript III First-Strand Synthesis SuperMix;

Invitrogen). Monkey MT2 melatonin receptor was then cloned using a nested PCR approach.


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The design of the specific primers for monkey MT2 melatonin receptor was based on rhesus

monkey genome data from the UCSC genome browser as well as on the monkey MT1

melatonin receptor. Using MT2-F1/R1 or MT2-F2/R2 primers (Table 1), the first and

second PCR amplifications were carried out in a total volume of 25 µl containing 0.5 µl of

Pfx50 DNA Polymerase, 2.5 µl of 10× Pfx50 PCR mix, 0.75 µl of 10 mM dNTPs, 2 µl of

3.75 µM each primer, and 2 µl of first strand cDNA or 1:400 dilution of the first-round PCR

products under the conditions described in Table 1.

DNA Sequencing

The final PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN)

and subcloned into a pCR-Blunt II-TOPO vector (Invitrogen). The clones were sequenced

in both directions using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems,

Foster City, CA) and an ABI Prism 3700 capillary sequencer.

Expression studies

The full-length monkey MT1 melatonin receptor cDNA was subcloned into the

expression vector pcDNA3.1 (Invitrogen). The myc peptide coding sequence was fused to

the C terminus of full-length and variants of the monkey MT2 receptor by PCR using

primers containing a linker and the myc sequence:

5′-CTAGTAAACGGCATGGCATCAATGCAGAAGCTGATCTCAGAGGAGGACCTGTA

G -3′. All plasmids were confirmed by sequencing analysis. CHO-K1 cells were cultured in

Eagle’s Minimum Essential Medium-α (MEM-α; Sigma-Aldrich) supplemented with 10%

fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) under

a 5% CO2/95% air atmosphere. For detection of myc-tagged monkey MT2 melatonin

receptor expression, CHO-K1 cells were transfected with pcDNA3.1 (C), or myc-tagged

full-length form (F) or a variant form (V1, V2, V3) of the monkey MT2 receptor expression

vector using Lipofectamine 2000 reagent (Invitrogen). At 5 h after transfection, the cells


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were treated with vehicle (DMSO), 2 μM MG-132 (Calbiochem Novabiochem Corporation,

La Jolla, CA), or 2 μM Cathepsin inhibitor I (Calbiochem Novabiochem) for 19 h. The cells

were then homogenized in lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25%

deoxycholic acid, 1% NP40, 1% SDS, 1 mM EDTA, and protease inhibitor cocktail

(Sigma-Aldrich). The insoluble component of the lysates was removed by centrifugation at

15000 × g for 20 min. Protein concentration was determined using a BCA protein assay kit

(Pierce, Hercules, CA). The lysates (20 μg of protein/lane) were subjected to SDS-PAGE

using Bis-Tris-glycine gels (4–12%) and transferred to polyvinylidene difluoride

membranes (Invitrogen). The membranes containing transferred proteins were probed with

a monoclonal anti-c-myc antibody (dilution 1:1000; Cell Signaling Technologies, Danvers,

MA).

Membrane preparation

CHO-K1 cells were transiently transfected with either monkey MT1 or MT2 receptor

(including variant forms) expression vector using Lipofectamine 2000 reagent (Invitrogen).

The transfected cells were washed with Dulbecco’s Phosphate-Buffered Saline (PBS;

Sigma-Aldrich) and then detached with PBS containing 1 mM EDTA. The cells were

pelleted by centrifugation at 1000 × g for 5 min, resuspended in ice-cold 50 mM Tris-HCl

buffer (pH 7.7 at 25°C), and then stored at –80°C until use. Brain membranes derived from

each region (SCN, cerebral cortex, putamen, caudate nucleus, amygdala) were isolated from

the brains of twelve cynomolgus monkeys. Crude membranes (CHO membranes, monkey

brain membranes) were prepared by homogenization in 50 mM Tris-HCl buffer (pH 7.7 at

25°C) and collected by two centrifugations at 40,000 × g for 20 min.

2-[125I] iodomelatonin binding studies

Saturation binding experiments were performed using 2-[125I] iodomelatonin. The

binding assay buffer contained CHO membranes or monkey SCN membranes diluted in 50


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mM Tris-HCl buffer (pH 7.7), melatonin (final conc. 1 or 10 µM) or vehicle (DMSO), and

increasing concentrations of 2-[125I] iodomelatonin (final conc. 6.25–800 pM) in a total

volume of 500 μL. After incubation for 120 min at 25°C, the reaction was terminated by the

addition of 4 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.7) followed by vacuum filtration

through a Whatman GF/B filter. The filter was washed twice and radioactivity was counted

using a γ-counter. Nonspecific binding was defined as the binding in the presence of 1 μM

(monkey brain membranes) or 10 μM melatonin (CHO membranes). Protein concentration

was determined using a BCA protein assay kit (Pierce, Rockford, IL). In order to compare

melatonin receptor densities across monkey brain regions (SCN, cerebral cortex, putamen,

caudate nucleus, amygdala), specific binding of 2-[125I] iodomelatonin was measured using

200 pM 2-[125I] iodomelatonin. Competitive binding experiments were performed using

2-[125I] iodomelatonin (CHO membranes: approximately 40 pM, monkey SCN membranes:

approximately 60 pM) with increasing concentrations (final conc. 0.64 pM–10 nM) of

unlabeled melatonin and ramelteon. Experiments were performed three times in CHO

membranes and once in monkey membranes from 12 monkeys due to limited availability of

tissue. Each concentration was assayed in duplicate.

cAMP studies

The receptor cDNAs were introduced into CHO-K1 cells using Lipofectamine 2000

reagent. Briefly, transfections were performed with the following DNA suspensions: 27.3

µg of monkey MT1 or MT2 receptor expression vector and 2.7 µg of human pituitary

adenylate cyclase-activating polypeptide receptor 1 (PAC1) expression vector in medium

containing 93.8 µl Lipofectamine 2000. At 24 h after transfection, the cells were washed

with PBS, and detached with PBS containing 1 mM EDTA. The cells were collected by

centrifugation at 1000 × g for 5 min, and then the cell pellets were resuspended in Hanks’

balanced salt solution containing 0.1% bovine serum albumin, 5 mM HEPES (pH 7.3), 0.5


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mM 3-isobutyl-1-methylxanthine (IBMX; Wako Pure Chemical Industries, Osaka, Japan).

The cells were stimulated with 1 nM pituitary adenylate cyclase-activating polypeptide

(PACAP27) in the presence of vehicle, melatonin, or ramelteon (0.26 pM–4 nM) for 20 min.

The amount of cAMP in the cells was measured using an AlphaScreen cAMP assay kit

(Perkin-Elmer, Wellesley, MA) according to the manufacturer’s protocol. Experiments were

performed three times in quadruplicate.

Data analysis

Equilibrium dissociation constants (Kd), receptor densities (Bmax), and IC50 values were

calculated from saturation and competition curves by nonlinear regression analysis using

PRISM software (GraphPad Software Inc., San Diego, CA). Data were fitted to a

three-parameter logistic model (binding studies), or a four-parameter logistic model (cAMP

studies) as a preferred model. Inhibition constants (Ki) were calculated from IC50 values

using the following equation:

Ki = IC50/(1 + L/Kd),

where L represents the concentration of 2-[125I] iodomelatonin in the binding assay.

The values (Kd, Ki, and IC50) for recombinant monkey melatonin receptors expressed in

CHO cells represent the mean ± S.E.M of three independent experiments.


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Results

Cloning of the monkey MT1 and MT2 melatonin receptors

We initially cloned the monkey MT1 and MT2 melatonin receptors by nested PCR

amplification of monkey SCN and eye mRNA-derived cDNAs, using exon 1- and exon

2-untranslated region primers based on an alignment of human melatonin receptor

sequences and the rhesus monkey genome sequence. Although monkey MT1 melatonin

receptor was not amplified from monkey SCN-derived cDNA using the nested PCR

approach, it was cloned from monkey genomic DNA (Fig. 1). The full-length monkey MT2

melatonin receptor and three novel splicing variant receptors (V1, V2, V3) were obtained

from monkey eye-derived cDNA (Figs. 2 and 3). The variant receptors had an additional

insertion between exon 1 and exon 2 of the monkey MT2 melatonin receptor gene. The

inserted sequences were found between exon 1 and exon 2 of rhesus monkey MT2

melatonin receptor genome sequence, and all of exon-intron splice junctions follow the

GT-AG rule. The insertions led to a frameshift in the coding sequence and the introduction

of a premature stop codon. The monkey MT1, MT2, and three variant (V1, V2, V3)

melatonin receptors encoded proteins of 352, 362, 88, 86, and 101 amino acids, respectively

(Figs. 1–3). The variant proteins consisted of 74 amino acids identical to the amino

terminus of the full-length monkey MT2 melatonin receptor, and unique carboxyl-terminal

amino acid sequences (Fig. 3). All the variants contained nonsense mutations in the coding

region of the deduced protein, and had only the first putative transmembrane domain.

Comparison of the amino acid sequences of MT1 melatonin receptors revealed that the

monkey MT1 melatonin receptor has the highest amino acid homology with the human MT1

melatonin receptor (95% identity) and has 85% and 84% identity to mouse and rat MT1

melatonin receptor, respectively (Fig. 1). Similarly, the monkey MT2 melatonin receptor

showed higher homology (96% identity) to the human MT2 melatonin receptor compared


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with mouse or rat MT2 melatonin receptor (Fig. 2). The amino acid sequence of monkey

melatonin receptors deduced from the rhesus monkey genome sequence was not fully

identical to those of the cloned receptors (MT1: a methionine to isoleucine exchange at

amino acid residue 203, MT2: an aspartate to asparagine exchange at amino acid residue 3).

Expression of the monkey MT2 variant receptors in CHO-K1 cells

To determine whether the monkey MT2 variant receptors have an ability to bind

melatonin receptor ligand, their binding properties were examined using CHO-K1 cells

transiently expressing the monkey MT2 receptor or variant receptors. The full-length

monkey MT2 melatonin receptor showed high specific binding to 2-[125I] iodomelatonin,

whereas none of the variant receptors did (Fig. 4). In order to confirm whether the variant

receptors were indeed translated in CHO-K1 cells, a myc tag was fused to the C terminus of

the full-length receptor (a positive control) and the variant receptors, and then their

expression levels were assessed by western blot analysis. A diffuse band of an

approximately 42-kDa protein representing myc-tagged full-length MT2 receptor protein

was expressed at a relatively high level (Fig. 5). The diffuse patterns of the full-length MT2

receptor and the human MT1 receptor proteins might be attributable to protein glcosylation

and high hydrophobicity (Brydon et al., 1999). In contrast, myc-tagged variant receptor

proteins (V1, V2, V3) were clearly detected only in the presence of the proteasome inhibitor

MG-132 (Fig. 5). Furthermore, two major bands were detected in the V3 variant lysates.

These results suggest that monkey MT2 variant receptor proteins were rapidly degraded by

proteasomes in CHO-K1 cells and also that the V3 variant receptor protein was cleaved by

an unknown mechanism.

Binding of 2-[125I] iodomelatonin to the recombinant and native monkey melatonin


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receptors

Previously, it was demonstrated that 2-[125I] iodomelatonin binds to both human MT1 and

MT2 melatonin receptors with high affinity (Reppert et al., 1994, 1995). We examined

whether monkey melatonin receptors had ligand binding characteristics similar to those of

human receptors by performing saturation binding experiments. Figure 6 shows saturation

curves of specific 2-[125I] iodomelatonin binding to CHO membranes expressing monkey

MT1 (Fig. 6A) or MT2 (Fig. 6B) receptors. For these receptors, 2-[125I] iodomelatonin

bound to a single class of high affinity binding sites. The equilibrium dissociation constant

(Kd) and maximum binding (Bmax) values (mean ± S.E.M; n = 3 experiments) were 19.9 ±

6.87 pM and 1.11 ± 0.33 pmol/mg protein, respectively, for the monkey MT1 receptor and

70.4 ± 7.05 pM and 1.35 ± 0.47 pmol/mg protein, respectively, for the monkey MT2

receptor. Thus, similar to the data previously published on the human MT1 and MT2

receptors, the monkey MT1 receptor had higher affinity for 2-[125I] iodomelatonin compared

to the monkey MT2 receptor (Kd = 21 ± 3 pM, 15 ± 3 pM for human MT1, and 107 ± 11 pM,

328 ± 12 pM for human MT2; Audinot et al., 2003, Kato et al., 2005, respectively). No

specific binding was detected in mock-transfected membranes (data not shown). We

proceeded to further characterize the 2-[125I] iodomelatonin binding sites in the monkey

SCN, which were expected to mediate the sleep-promoting effects of melatonin and

ramelteon. Autoradiographic studies with 2-[125I] iodomelatonin have revealed the presence

of high affinity binding sites in the rhesus monkey SCN (Weaver et al., 1993). The monkey

SCN membranes specifically bound 2-[125I] iodomelatonin with 4-times lower affinity (Fig.

6C; Kd = 80.5 pM) than that of the recombinant monkey MT1 melatonin receptor. In

addition, the amount of specific 2-[125I] iodomelatonin binding in SCN membrane was

higher than that in membranes from other regions of the monkey brain (SCN, 0.466

fmol/mg protein; cerebral cortex, 0.005 fmol/mg protein; putamen, 0.145 fmol/mg protein;


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caudate nucleus, 0.173 fmol/mg protein; amygdala, 0.106 fmol/mg protein).

Competition by ramelteon and melatonin for 2-[125I] iodomelatonin binding to the

recombinant and native monkey melatonin receptors

We determined the affinities of ramelteon and melatonin for recombinant and native

monkey melatonin receptors by performing competition binding experiments. Figure 7

illustrates the concentration-dependent inhibition of 2-[125I] iodomelatonin binding to

recombinant (Fig. 7A, 7B) and native monkey melatonin receptors (Fig. 7C) by ramelteon

and melatonin (0.64 pM–10 nM). Ramelteon displayed 3- to 7-fold higher affinities for

recombinant and native monkey melatonin receptors than melatonin, which is consistent

with the results for human melatonin receptors (Table 2). Nevertheless, although the affinity

of ramelteon for monkey MT1 melatonin receptor was comparable to that for human MT1

melatonin receptor, its affinity for monkey MT2 melatonin receptor was higher than that for

human MT2 melatonin receptor (Ki = 14 ± 0.5 pM and 112 ± 5 pM for the human MT1

and MT2 melatonin receptors, respectively; Kato et al., 2005).

Inhibition of PACAP27-stimulated cAMP production in CHO cells transiently

coexpressing human PAC-1 with the monkey melatonin receptors.

In order to confirm that the recombinant monkey melatonin receptors are functionally

coupled to adenylate cyclase, we examined the effects of ramelteon and melatonin on

PACAP27-stimulated cAMP production in CHO-K1 cells transiently coexpressing human

PAC-1 with the monkey MT1 or MT2 melatonin receptors. Previous studies have shown that

melatonin suppresses PACAP-induced phosphorylation of Ca2+/cAMP response element

binding protein (CREB) in mice (von Gall et al., 2000). Ramelteon and melatonin caused a

dose-dependent inhibition of the cAMP production induced by PACAP27 (Fig. 8A, 8B). For


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ramelteon, the IC50 values (n = 3) were 28.5 ± 8.55 pM and 20.1 ± 9.25 pM for the monkey

MT1 and MT2 receptors, respectively, whereas the corresponding values for melatonin were

274 ± 40.2 pM and 111 ± 24.6 pM. Thus, the agonist activity of ramelteon at monkey MT1

and MT2 receptors was 9.6 and 5.5 times more potent, respectively, than melatonin.


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Discussion

In this study, we characterized the cynomolgus monkey MT1 and MT2 melatonin

receptors, and determined the in vitro activity of ramelteon at these receptors as compared

with melatonin. Ramelteon displayed higher affinities than melatonin for monkey

recombinant and native melatonin receptors; moreover, these affinities for monkey

receptors were comparable to those for human receptors (Fig. 2; Kato et al., 2005).

Furthermore, ramelteon inhibited PACAP27-induced cAMP production with a higher

potency than melatonin. These observations indicate that the pharmacological profiles of

the monkey melatonin receptors are similar to those of the human melatonin receptors.

We initially cloned the novel genes for cynomolgus monkey MT1 and MT2 melatonin

receptors, and also three MT2 splice variants. The monkey MT1 and MT2 melatonin

receptors had significant amino acid homology with the human MT1 and MT2 melatonin

receptors, i.e., 95% and 96% identity, respectively. In addition, the monkey melatonin

receptors showed high amino acid homology with rat (84% for MT1, 80% for MT2,

respectively) and mouse homologues (85% for MT1, 80% for MT2, respectively).

Site-directed mutagenesis studies dealing with melatonin receptors have demonstrated that

some amino acid residues are critical for ligand binding. Two serine residues (Ser110 and

Ser114) in transmembrane domain 3 (TM3) of human MT1 receptor were required for

agonist binding (Conway et al., 2001). Furthermore, a valine residue and a histidine residue

in the TM5 of ovine melatonin receptor (corresponding to Val192 and His195 in the human

MT1 receptor) were associated with the binding affinities and potencies of ligands (Conway

et al., 1997). On the basis of a 3-D model of human MT2 receptor obtained using the X-ray

structure of bovine rhodopsin as a template, Mazna et al. (2004) reported that a valine

(Val204) residue in TM5, a leucine (Leu272) residue in TM6, and a tyrosine (Tyr298) residue

in TM7 were involved in 2-iodomelatonin binding. These amino acid residues are


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conserved in the respective receptors of the 4 species mentioned above (Figs. 1 and 2). In

accord with these high amino acid identities, the Kd and Ki values (ramelteon and

melatonin) for the monkey melatonin receptors, especially MT1 receptor, were comparable

to those for the human melatonin receptors expressed in the same cell line of CHO cells

(Audinot et al., 2003, Kato et al., 2005). The monkey MT2 variant receptors each contained

different new exons between exon 1 and exon 2. Both human MT1 and MT2 melatonin

receptor genes contain a conserved intron splice site in the first cytoplasmic loop. This

intron could lead to alternative splicing forms of these receptors (Reppert et al., 1995).

Indeed, Slominski et al. (2003) reported that the human MT2 variant receptor had an

insertion of 154 bp from intron 1 and lacked a 200 bp from exon 2, which generated a

frameshift and premature translation termination; the resultant truncated protein consisted

of 73 N-terminal amino acids. The Siberian hamster MT2 receptor lacks exon 1, and also

has two nonsense mutations in the coding region (Weaver et al., 1996). Similar to the

monkey MT2 variant receptors, it is unlikely that such variants are functional.

We demonstrated the binding profiles of the native monkey melatonin receptor in the

cynomolgus monkey SCN. High affinity binding sites of 2-[125I] iodomelatonin are

observed in the SCN of humans (Kd, 53.3 pM), rhesus monkeys (Weaver et al., 1993), and

rats (Kd, 52.8 pM; Laitinen and Saavedra, 1990). The binding affinity at monkey SCN

membranes (Kd, 80.5 pM, Fig. 6C) is consistent with these reports, whereas the Kd value for

the monkey SCN membranes was different to that for either of the cloned monkey

melatonin receptors. It remains unclear whether the MT2 melatonin receptor is expressed in

the monkey SCN. Consistent with mouse and rat SCN, the majority of 2-[125I]

iodomelatonin binding in the monkey SCN may be due to the presence of the MT1

melatonin receptor (Liu et al., 1997; Dubocovich et al., 1998).

For functional studies, we used CHO-K1 cells transiently coexpressing human PAC-1


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with the monkey MT1 or MT2 melatonin receptors. In order to increase the maximum

potency of melatonin, which inhibits PACAP27-stimulated cAMP production, the ratio of

amounts of PAC-1 vector to either the MT1 or MT2 melatonin receptor was approximately

1/10. Melatonin inhibits the PACAP-induced phosphorylation of CREB in the mouse SCN

mediated by MT1 melatonin receptor and presumably MT2 melatonin receptor (von Gall et

al., 1998). The cotransfected cells may mimic cAMP signaling in the SCN cells. For both

the monkey MT1 and MT2 melatonin receptors, ramelteon was shown to be an agonist with

a higher potency than melatonin. Activation of the monkey MT1 and MT2 melatonin

receptors by ramelteon and melatonin, as determined by the inhibition of

PACAP27-stimulated cAMP production, was correlated with their binding affinities. For

example, ramelteon exhibited a 3.2-fold higher affinity and 5.5-fold higher potency than

melatonin at the monkey MT2 melatonin receptor. In contrast, the relative potency of

ramelteon and melatonin at the human MT2 melatonin receptor differs considerably from

that predicted by binding activity (Kato et al., 2005). Melatonin has a 17-fold lower potency

than ramelteon compared with a 3.4-fold lower affinity at the human MT2 receptor. The

species differences in MT2 melatonin receptor sequence may be involved in this

phenomenon.

In conclusion, this study provides new information on the characters of native and

recombinant monkey melatonin receptors. Similar to human homologues, ramelteon

exhibited higher binding affinities and more potent functional activities than melatonin at

monkey melatonin receptors. This information on monkey melatonin receptors will help us

to gain an understanding of the pharmacological effects (e.g., sleep promotion) of melatonin

receptor agonists in monkeys. In addition, the sequence and functional similarities between

the melatonin receptors of monkeys and humans suggest that an evaluation of

sleep-promoting effect of melatonin receptor agonists in monkeys may be useful for


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predicting the efficacy of such agonists in humans.


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Acknowledgements

We wish to thank Margarita L. Dubocovich, Hideaki Nagaya, and Takeo Wada for

critical reading of the manuscript and valuable comments; Hisao Nishikawa, Yoshiyuki

Furukawa, Eiji Nishida, and Ryoetsu Imai for collecting monkey tissues; Minoru Maruyama

for donating the PACAP 1 receptor expression vector; and Michiyasu Takeyama and Yugo

Habata for useful technical suggestions.


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Footnotes

Reprint Requests: Shin-ichi Yoshikubo, Ph.D., Pharmacology Research Laboratory,

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 17-85,

Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan

Phone: +81-6-6300-6647, Fax: +81-6-6300-6306, E-mail:

[email protected]


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Legends for figures

Fig. 1. Comparison of the amino acid sequences of monkey, human, mouse, and rat MT1

melatonin receptors. Consensus amino acid residues in more than three receptors are boxed.

Gaps in the sequences are represented by dashes. The putative seven transmembrane

domains are overlined. The DNA sequences for monkey, human, mouse, and rat MT1

melatonin receptors have been deposited in GenBank under the accession numbers

FJ918400, NM_005958, NM_008639, and NM_053676, respectively.

Fig. 2. Comparison of the amino acid sequences of monkey, human, mouse, and rat MT2

melatonin receptors. Consensus amino acid residues in more than three receptors are boxed.

Gaps in the sequences are represented by dashes. The putative seven transmembrane

domains are overlined. The DNA sequences for monkey, human, mouse, and rat MT2

melatonin receptors have been deposited in GenBank under the accession numbers

FJ905899, NM_005959, NM_145712, and NM_001100641, respectively.

Fig. 3. Comparison of the amino acid sequences of monkey full-length MT2 melatonin

receptor and three types of MT2 splicing variant (variants 1–3). Consensus amino acid

residues in more than three receptors are boxed. The putative seven transmembrane

domains are overlined. The DNA sequences for monkey MT2 splicing variants have been

deposited in GenBank under the accession numbers FJ905900, FJ905901, and FJ905902,

respectively.

Fig. 4. Specific binding of 2-[125I] iodomelatonin to vehicle or 2 μM MG-132-treated CHO

membranes expressing full-length (F) or variant forms (V1, V2, V3) of monkey MT2

receptor. Data shown are mean ± S.E.M. of three experiments.


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Fig. 5. Rapid degradation of monkey MT2 variant receptor proteins by proteasomes.

CHO-K1 cells were transiently transfected with control vector (C), myc-tagged full-length

form (F), or variant forms (V1, V2, V3) of monkey MT2 receptor expression vectors, and

then treated with vehicle (lanes 1 to 5), 2 μM MG-132 (lanes 6 to 9); or 2 μM cathepsin

inhibitor I (lanes 10 to 13) for 19 h. Cells were harvested for western blotting of

myc-tagged protein. Although the expression of myc-tagged full-length MT2 receptor

proteins was verified, MT2 variant receptor proteins were clearly detected only after

MG-132 treatment. Molecular weights in kilodaltons (kDa), determined from prestained

standards, are indicated on the left.

Fig. 6. Saturation binding of 2-[125I] iodomelatonin to CHO membranes expressing monkey

MT1 (A) or MT2 (B) receptors and monkey suprachiasmatic nucleus (SCN) membranes (C).

(Inset) Scatchard plot of the saturation data. Data shown are for representative experiments

with each point representing duplicate determinations.

Fig. 7. Competition by ramelteon and melatonin for 2-[125I] iodomelatonin binding to CHO

membranes expressing monkey MT1 (A) or MT2 (B) receptors and monkey suprachiasmatic

nucleus (SCN) membranes (C). Values are expressed as a percentage of specific binding.

Data shown are mean ± S.E.M. of three (A, B) experiments or from a single (C) experiment,

with each point representing duplicate determinations. Ki values are listed in Table 2.

Fig. 8. Inhibition of PACAP27-stimulated cAMP production in CHO cells transiently

coexpressing human PACAP receptor 1 (PAC-1) with monkey MT1 (A) or MT2 (B) receptor.

The 100% value is the mean cAMP production induced with 1 nM PACAP27. Data shown

are mean ± S.E.M. of three experiments with each point representing quadruplicate


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determinations.


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Table 1. Primer sequences and conditions used for PCR amplification

Primer name Sequence (5′-3′) Conditions

Primers for MT1

MT1-exon1-F1 ACACGTGAATGAACAGCCTCGCTG 95°C × 3 min, 1 cycle / 95°C × 30 s, 61.8°C × 30 s,

MT1-exon1-R1 GTCCCCATAGGAAAGAGAATGCGACTCT 72°C × 30 s, 25 cycles / 72°C × 10 min, 1 cycle

MT1-exon2-F1 GCAGCTGTGACGTCAGGTCATCAG 94°C × 2 min, 1 cycle / 94°C × 15 s, 60°C × 20 s,

MT1-exon2-R1 AAGCTGGAAGGTTCCTCCACCA 68°C × 90 s, 20 cycles / 68°C × 5 min, 1 cycle

MT1-exon1-F2 AAGCGGGCTCGCGACGGA 95°C × 3 min, 1 cycle / 95°C × 30 s, 61.8°C × 30 s,

MT1-exon1-R2 CGATGGGCAGGCTGGAGGAAGA 72°C × 30 s, 25 cycles / 72°C × 10 min, 1 cycle

MT1-exon2-F2 GCAGGCAGAAGCAGCTCCTTCTT 94°C × 2 min, 1 cycle / 94°C × 15 s, 60°C × 20 s,

MT1-exon2-R2 AGGCAACACGGAAAGGCGTG 68°C × 90 s, 30 cycles / 68°C × 5 min, 1 cycle

MT1-exon1-F3 ATGCCGGGCAACGGCAGCGCGCTGC 95°C × 3 min, 1 cycle / 95°C × 30 s, 55°C × 30 s,

MT1-exon1-R3 AAGATGTTTCCTGCGTTCCGGAGCTTCTTGTTCC

G 72°C × 30 s, 30 cycles / 72°C × 10 min, 1 cycle

MT1-exon2-F3 CGGAACGCAGGAAACATCTTTGTGGTGAGCTTA

GC 94°C × 2 min, 1 cycle / 94°C × 15 s, 60°C × 20 s,

MT1-exon2-R3 TTAAACCGAGTCCACCTTTACTAGA 68°C × 1 min, 20 cycles / 68°C × 5 min, 1 cycle

MT1-exon1-F3 95°C × 3 min, 1 cycle / 95°C × 30 s, 55°C × 30 s,

MT1-exon2-R3 72°C × 30 s, 30 cycles / 72°C × 10 min, 1 cycle

Primers for MT2

MT2-F1 CAGTACTGCGCGAGCCCTGCG 94°C × 2 min, 1 cycle / 94°C × 15 s, 68.4°C × 20 s,

MT2-R1 CACCATTTCATGAGTTTGTCGTGC 68°C × 90 s, 20 cycles / 68°C × 5 min, 1 cycle

MT2-F2 AAAGCGCAGCGCGGGAGAGT 94°C × 2 min, 1 cycle / 94°C × 15 s, 66.8°C × 20 s,

MT2-R2 AGCTGGTGTGCCTCAGATCCAG 68°C × 90 s, 30 cycles / 68°C × 5 min, 1 cycle


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Table 2. Affinities of ramelteon and melatonin for native and recombinant monkey

melatonin receptors. The inhibition constants (Ki) of ramelteon and melatonin were

calculated from IC50 and Kd using the Cheng-Prusoff equation. Values for recombinant

receptors represent the mean ± S.E.M. of three independent experiments.

Ki (pM)

Compound Monkey MT1 (CHO) Monkey MT2 (CHO) Monkey SCN

Ramelteon 12.3 ± 1.00 40.4 ± 2.86 49.4

Melatonin 67.4 ± 24.3 129 ± 16.8 329


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Molecular Cloning and Pharmacological Characterization of … · 2008. 12. 31. · respectively. Saturation binding experiments with 2-[125I] iodomelatonin revealed that the dissociation