Elsevier

Biochemical Pharmacology

Volume 75, Issue 10, 15 May 2008, Pages 2007-2019
Biochemical Pharmacology

Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors

https://doi.org/10.1016/j.bcp.2008.02.022Get rights and content

Abstract

In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT1 receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT2 receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT2 receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT1 and rMT2. A 55% homology was observed between both rat isoforms. The entire contiguous rat MT1 and MT2 receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[125I]-Iodomelatonin. The dissociation constants (Kd) for rMT1 and rMT2 were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT1 and MT2 receptors were evaluated at rMT1 and rMT2 receptors, for their binding affinity and functionality in [35S]-GTPγS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT1 subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

Introduction

The indole hormone melatonin was first discovered in the bovine pineal gland [1], and was subsequently found in a large variety of vertebrates species [2]. Melatonin is synthesized and released in a circadian fashion by the pineal gland and has been shown to play a modulatory role in diverse physiological functions including circadian entrainment, reproduction, sleep and blood pressure [2], [3], [4]. Two mammalian receptors have been cloned. The MT1 and MT2 receptors are G-protein coupled receptors that exhibit sub-nanomolar affinity for melatonin [5], [6], [7]. A third melatonin binding site named MT3 has a 10 nM range affinity for melatonin and was described in hamster as the human homologue of the cytoplasmic quinone reductase 2 [8], [9]. In order to assign specific roles for each subtype, pharmacological tools are needed [10]. There are only a few ligands selective for one melatonin receptor subtype: Luzindole, 4P-PDOT, S 24773 and S 24014 are selective for the MT2 melatonin receptor subtype [11], [12], [13], [14], [15], [16], whereas S 26131 and S 26284 are selective for MT1[17]. We have recently published the characterization of several different compounds distinguishing human MT1 and MT2 receptors expressed in CHO cells [17]. All these affinity data have been obtained on human receptors whereas in vivo experiments are usually performed on rats. We wondered whether the data found on human recombinant receptors could be predictive or not to that at the rat recombinant receptors. No systematic survey or characterization of melatonin binding at recombinant receptors from different species is available yet, apart from our work on human and ovine species [17], [18]. Since there is yet no published data describing the rat melatonin receptors either at the molecular level or at the pharmacological level, we undertook this study in which we systematically compared the rat and human MT1 and MT2 receptors.

Section snippets

Reagents and ligands

The two radioligands (2-[125I]-iodomelatonin (specific activity: 2000 Ci/mmol) and [35S]-GTPγS (Guanosine-5′[γ-35S]-triphosphate; specific activity: 1000 Ci/mmol) were purchased from Perkin-Elmer. Melatonin and 2-iodomelatonin were obtained from Sigma and 4P-PDOT (4-phenyl-2-propionamidotetraline), luzindole (2-benzyl-N-acetyltryptamine), 6-chloromelatonin from Tocris. Fourteen analogues of melatonin were evaluated (Fig. 1). Their synthesis were reported in the following reports, mostly patents:

Rat melatonin receptors

Due to limited distribution and low abundance, rat MT1 and MT2 melatonin receptors were cloned using a strategy of screening a rat genomic library for exon 1 and exon 2 followed by ligation of appropriate exons to complete a full length receptor clone (Fig. 2, Fig. 3). Our cloned full length rat MT1 receptor comprises 353 amino acid in length and found to be identical to a predicted rat MT1 receptor (Genbank number XP_341442) except for 1 amino acid at position 270, (T270A) located in the 2nd

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