Characterization and regulation of the human ML(1A) melatonin receptor stably expressed in Chinese hamster ovary cells

The human ML(1A) melatonin receptor is expressed in the suprachiasmatic nucleus of the hypothalamus and is believed to regulate circadian rhythms. We report the kinetic characteristics and pharmacological profile of 2- [¹²⁵I]iodomelatonin binding and the signaling pathway and agonist regulation of the human ML(1A) melatonin receptor stably expressed in Chinese hamster ovary cells. Association of 2-[¹²⁵I]iodomelatonin binding was maximal by 1.5 hr at 37° and fully dissociated on the addition of 1 μM melatonin. The binding of 2-[¹²⁵I]iodomelatonin was saturable and of high affinity (K(D) = 74 ± 14 pM, B(max) = 679 ± 88 fmol/mg protein; three experiments). The pharmacological profile of various melatonin analogues revealed a profile (2-iodomelatonin ≤ melatonin > N-acetyl serotonin > luzindole) characteristic of an ML₁ subtype. Competition of melatonin for 2- [¹²⁵I]iodomelatonin binding to the human ML(1A) receptor in lysed or intact cells resulted in biphasic curves revealing the existence of super high (~20%) and high (~80%) affinity states of the receptor. Guanosine-5'- O-(3-thio)triphosphate (100 pM-30 μM) when added alone inhibited 2- [¹²⁵I]iodomelatonin binding (IC₅₀ = 0.87 ± 0.12 μM; three experiments), suggesting uncoupling of the receptor from G proteins. In addition, guanosine-5'-O-(3-thio)triphosphate (3 μM) produced a right-ward shift in both the super high and high binding melatonin affinities for 2- [¹²⁵I]iodomelatonin resulting in monophasic curves. Melatonin (0.1 fM-1 nM) inhibited forskolin-induced cAMP formation in a concentration-dependent and biphasic manner. Low concentrations of melatonin (0.01 fM-1 pM) inhibited forskolin (100 μM)-stimulated cAMP formation with an IC₅₀ of 0.1 ± 0.05 pM (four experiments) and a maximal inhibitory effect (26%) at 1 pM. Higher concentrations of melatonin (1 pM-1 nM) inhibited forskolin-induced cAMP formation with an IC₅₀ of 64 ± 1.8 pM (four experiments) and a maximal inhibition (74%) at 1 nM. Luzindole (1 μM), a competitive melatonin receptor antagonist, antagonized the effect of melatonin at the higher concentrations only (IC₅₀ = 1.5 ± 0.22 nM, pK(B) = 7.3; three experiments). Pretreatment with pertussis toxin completely abolished melatonin-mediated inhibition of forskolin-induced cAMP formation through these receptors. Pretreatment with various concentrations of melatonin (0.1 pM-1 μM) for different periods of time (1, 6, 18, and 24 hr) did not decrease 2-[¹²⁵I]iodomelatonin binding. However, competition by melatonin for 2-[¹²⁵I]iodomelatonin binding to cells pretreated with melatonin and washed was only to a single population of super high affinity sites (IC₅₀ = 1.1 ± 0.28 nM; three experiments) as revealed by monophasic curves. Cells pretreated with melatonin revealed a persistent inhibition (~20%) of forskolin-induced cAMP formation that was not reversed by extensive washes (up to 1 hr) or when luzindole (1 μM) was added together with melatonin during pretreatment. These results suggest that tight binding of melatonin to the super high affinity state of the human ML(1A) melatonin receptor may be the mechanism by which low concentrations of circulating hormone in vivo regulates signaling in the suprachiasmatic nucleus of the hypothalamus.