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 suprachiasmati c nucleus of the hypothalamus and is believed to regulate circadian rh ythms. We report the kinetic characteristics and pharmacological profi le of 2-[I-125]iodomelatonin binding and the signaling pathway and ago nist regulation of the human ML(1A) melatonin receptor stably expresse d in Chinese hamster ovary cells. Association of 2-[I-125]iodomelatoni n binding was maximal by 1.5 hr at 37 degrees acid fully dissociated o n the addition of 1 mu M melatonin. The binding of 2-[I-125]iodomelato nin was saturable and of high affinity (K-D = 74 +/- 14 pM, B-max = 67 9 +/- 88 fmol/mg protein; three experiments). The pharmacological prof ile of various melatonin analogues revealed a profile (2-iodomelatonin greater than or equal to melatonin > N-acetyl serotonin > luzindole) characteristic of an ML(1) subtype. Competition of melatonin for 2-[I- 125]iodomelatonin binding to the human ML(1A) receptor in lysed or int act cells resulted in biphasic curves revealing the existence of super high (similar to 20%) and high (similar to 80%) affinity states of th e receptor. Guanosine-5'-O-(3-thio)triphosphate (100 pM-30 mu M) when added alone inhibited 2-[I-125]iodomelatonin binding (IC50 = 0.87 +/- 0.12 mu M; three experiments), suggesting uncoupling of the receptor f rom G proteins. In addition, guanosine-5'-O-13-thio)triphosphate (3 mu M) produced a rightward shift in both the super high and high binding melatonin affinities for 2-[I-125]iodomelatonin resulting in monophas ic curves. Melatonin (0.1 fM-1 nM) inhibited forskolin-induced cAMP fo rmation in a concentration-dependent and biphasic manner. Low concentr ations of melatonin (0.01 fM-1 pM) inhibited forskolin (100 mu M)-stim ulated cAMP formation with an IC50 of 0.1 +/- 0.05 pM (four experiment s) and a maximal inhibitory effect (26%) at 1 pM. Higher concentration s of melatonin (1 pM-1 nM) inhibited forskolin-induced cAMP formation with an IC50 of 64 +/- 1.8 pM (four experiments) and a maximal inhibit ion (74%) at 1 nM. Luzindole (1 mu M), a competitive melatonin recepto r antagonist, antagonized the effect of melatonin at the higher concen trations only (IC50 = 1.5 +/- 0.22 nM, pK(B) = -7.3; three experiments ). Pretreatment with pertussis toxin completely abolished melatonin-me diated inhibition of forskolin-induced cAMP formation through these re ceptors. Pretreatment with various concentrations of melatonin (0.1 pM -1 mu M) for different periods of time (1, 6, 18, and 24 hr) did not d ecrease 2-[I-125]iodomelatonin binding. However, competition by melato nin for 2-[I-125]iodomelatonin binding to cells pretreated with melato nin and washed was only to a single population of super high affinity sites (IC50 = 1.1 +/- 0.28 nM; three experiments) as revealed by monop hasic curves. Cells pretreated with melatonin revealed a persistent in hibition (similar to 20%) of forskolin-induced cAMP formation that was not reversed by extensive washes (up to 1 hr) or when luzindole (1 mu M) was added together with melatonin during pretreatment. These resul ts suggest that tight binding of melatonin to the super high affinity state of the human ML(1A) melatonin receptor may be the mechanism by w hich low concentrations of circulating hormone in vivo regulates signa ling in the suprachiasmatic nucleus of the hypothalamus.