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Novel Isoforms of Mel_1c Melatonin Receptors Modulating Intracellular Cyclic Guanosine 3',5'-Monophosphate Levels

CNRS-UPR 0415 and Université Paris VII (R.J., L.P., I.L., P.C., S.M., A.D.S.) Institut Cochin de Génétique Moléculaire F-75014 Paris, France
Molecular Neuroendocrinology Group (P.B., P.J.M.) Rowett Research Institute Aberdeen, AB2 9SB UK
Institut de Recherches Internationales Servier (B.G., P.D), F-92415 Courbevoie Cedex, France

Two cDNAs encoding novel isoforms of Xenopus laevis melatonin receptors were cloned using PCR primers specific for the X. laevis-melanophore Mel_1c melatonin receptor described in a recent publication. The novel isoforms were highly homologous to the described frog Mel_1c cDNA, although the C-terminal tail of both was shorter by 65 amino acid residues. Nucleotide sequences of these novel isoforms, called Mel_1c() and Mel_1c(ß), differed from each other by only 35 nucleotides and six amino acid residues. Studies on several animals of various Xenopus species indicate that Mel_1c() and Mel_1c(ß) receptors may correspond to allelic variants of the same locus.

Studies on cells transfected with both receptor cDNAs showed the expression of high-affinity 2-[¹²⁵I]iodomelatonin binding sites. Agonist stimulation of Mel_1c() receptor was associated with the inhibition of cAMP accumulation stimulated by forskolin (IC₅₀ 10^-10 M) in HeLa, Ltk^-, and human embryonic kidney 293 (HEK 293) cells. Mel_1c(ß) receptor modulated cAMP in HeLa and HEK 293 cells but not in Ltk^- cells. Both receptors inhibited, in a dose-dependent manner, cGMP accumulation in all three cell lines incubated with a phosphodiesterase inhibitor. This effect was localized upstream of soluble guanylyl cyclase and was blocked by pertussis toxin treatment. However, IC₅₀ values (10^-10 M for Mel_1c(ß) and 10^-9 to 10^-7 M for Mel_1c()) and maximal inhibition levels showed that Mel_1c() receptors are much less efficiently coupled to the cGMP pathway.

Coupling differences may be explained by the fact that five of the six amino acid substitutions between Mel_1c() and Mel_1c(ß) receptors are located within cytoplasmic regions potentially involved in signal transduction. The existence of coupling differences is in agreement with the observation that expression of both receptors is evolutionally conserved in native tissue. In conclusion, two novel, potentially allelic, isoforms of Xenopus Mel_1c melatonin receptors display identical ligand-binding characteristics, but different potencies in modulating cAMP and cGMP levels through G_i/G_o-dependent pathways. Furthermore, to our knowledge, this study provides the first data on the modulation of intracellular cGMP levels by cloned melatonin receptors.

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