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