Ebisawa T et al. (1994), Expression cloning of a high-affinity melatonin...

XB-ART-21139

Proc Natl Acad Sci U S A 1994 Jun 21;9113:6133-7.

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Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

Ebisawa T , Karne S , Lerner MR , Reppert SM .

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Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.

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Species referenced: Xenopus laevis
Genes referenced: camp mtnr1al mtnr1c

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	FIG. 1. Expression of the Xenopus melatonin receptor cDNA in COS-7 cells assayed by 125I-Mel binding. (Upper) Saturation curve. Nonspecific binding was determined by using 10 MM melatonin. (Lower) Scatchard plot of saturation data. Data shown are representative of three experiments.
	FIG. 2. Competition by various ligands for 125I-Mel binding in COS-7 cells transfected with the melatonin receptor cDNA. Cells were incubated with 100 pM 125I-Mel and various concentrations of 2-iodomelatonin (I-Mel), melatonin (Mel), 6-chloromelatonin (6C1- Mel), 6-hydroxymelatonin (60H-Mel), N-acetyl-5-hydroxytryptamine (NAS), or 5-hydroxytryptamine (SIT). Nonspecific binding was determined in the presence of 10MuM melatonin. Ki values are as follows: I-Mel, 1.1 x 10-10 M; Mel, 1.3 x 10-9 M; 6C1-Mel, 3.0 x 10-9 M; 60H-Mel, 2.0 x 10-8 M; NAS, 2.0 x 10-6 M; 5HT, >1.0 X 10-4 M. Inhibition curves were generated by LIGAND using a one-site model. Data shown are representative of three experiments.
	FIG. 3. Melatonin inhibition of forskolin-stimulated cAMP accumulation in CHO cells stably transfected with melatonin receptor cDNA. The 100%1 value is the mean cAMP value induced with 10 PM forskolin. Data shown are representative of three experiments.
	FIG. 4. Northern blot analysis of melatonin receptor transcripts in Xenopus dermal melanophores. Lane contained 3 jg of poly(A)+ RNA. Locations ofRNA size markers (GIBCO/BRL) are indicated. Blot was exposed to x-ray film overnight.
	FiG. 5. Proposed membrane structure of the Xenopus melatonin receptor. Deduced amino acid sequence is shown. Yt, Potential N-linked glycosylation site. Solid circles, consensus sites for protein kinase C phosphorylation.

References [+] :

Abe, Role of cyclic AMP in mediating the effects of MSH, norepinephrine, and melatonin on frog skin color. 1969, Pubmed