Elsevier

Biochemical Pharmacology

Volume 58, Issue 4, 15 August 1999, Pages 633-639
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3′-5′-monophosphate pathway

https://doi.org/10.1016/S0006-2952(99)00134-3Get rights and content

Abstract

Cyclic guanosine 3′-5′-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3′-5′-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.

Section snippets

Cloning and expression of human Mel1a and Mel1b receptors

The human Mel1a receptor cDNA was cloned by RT-PCR from human embryonic RNA and by PCR from human genomic DNA. Human embryonic brain RNA was reverse transcripted as described [5]. Mel1a-specific primers were selected from the published sequence [8]. DNAs were amplified with high-fidelity Pfu or Pwo DNA polymerase as described previously [5]. The N-terminal part corresponding to the first exon was amplified using PCR primers 23S(5′-AACAAAGCTTATGCAGGGC-3′) and 203AS (5′-GATGTTGCCGGCGTTCCTGAGC)

Cloning, expression, and pharmacological characterization of human Mel1a and Mel1b receptors

The coding region and the 3′ untranslated region of the Mel1a receptor were cloned by RT-PCR from human embryonic brain RNA. Mel1a-specific PCR primers were selected from the recently published sequence [8]. The human Mel1b receptor gene was isolated from human genomic DNA by PCR using Mel1b-specific primers selected from the recently published sequence [4]. Both receptor DNAs were cloned into the expression vector pcDNA3/RSV and their identity was confirmed by sequencing. No amino acid

Discussion

Cyclic GMP is an important second messenger in the central nervous system [22], in blood vessels, and in the retina, locations where melatonin receptors are expressed 4, 6, 23, 24. Melatonin is secreted from the pineal gland in a circadian manner with maximal levels during the night. Cyclic cGMP levels have also been shown to vary in a circadian manner in the cerebral cortex of the chick, where they are correlated to blood rhythms of melatonin [25]. Interestingly, the cycle of cGMP and

Acknowledgements

This work was supported by grants from CNRS, the Association pour la Recherche sur le Cancer (N°9890), the University of Paris, and the Intitut de Recherches Internationales Servier (IRIS). We thank Drs. S. Marullo, P. J. Morgan, and P. Barret for their expert advice and help in the initial phase of receptor cloning.

References (32)

  • A Cagnacci

    Melatonin in relation to physiology in adult humans

    J Pineal Res

    (1996)
  • M Viswanathan et al.

    Expression of melatonin receptors in arteries involved in thermoregulation

    Proc Natl Acad Sci USA

    (1990)
  • S.M Reppert

    Melatonin receptorsMolecular biology of a new family of G protein-coupled receptors

    J Biol Rhythms

    (1997)
  • S.M Reppert et al.

    Molecular characterization of a second melatonin receptor expressed in human retina and brainThe Mel1b melatonin receptor

    Proc Natl Acad Sci USA

    (1995)
  • R Jockers et al.

    Novel isoforms of Mel1c melatonin receptor modulating intracellular cyclic guanosine 3′-5′-monophosphate levels

    Mol Endocrinol

    (1997)
  • C Mazzucchelli et al.

    Expression of the melatonin receptor in Xenopus LaevisA comparative study between protein and mRNA distribution

    J Pineal Res

    (1996)
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