Characterization and regulation of the human ML1A melatonin receptor stably expressed in Chinese hamster ovary cells.
Characterization and regulation of the human ML1A melatonin receptor stably expressed in Chinese hamster ovary cells.
复制标题
DOI：
--
复制
发表时间：
1996-07
影响因子：
3.6
通讯作者：
P. Witt‐Enderby;M. Dubocovich
中科院分区：
文献类型：
--
作者：
P. Witt‐Enderby;M. Dubocovich
人ML 1A褪黑激素受体在下丘脑的视交叉上核中表达，并被认为调节昼夜节律。我们报告的动力学特征和药理学概况2-[125 I]碘褪黑素结合和信号转导途径和激动剂调节的人ML 1A褪黑素受体在中国仓鼠卵巢细胞中稳定表达。协会的2-[125 I]碘褪黑激素的结合是最大的1.5小时，在37度和完全解离的添加1 μ M褪黑激素。2-[125 I]碘褪黑激素的结合是可饱和的并且具有高亲和力（KD = 74 +/- 14 PM，Bmax = 679 +/- 88 fmol/mg蛋白;三个实验）。各种褪黑激素类似物的药理学特征揭示了ML 1亚型的特征（2-碘褪黑激素>或=褪黑激素> N-乙酰5-羟色胺>卢吲哚）。褪黑激素竞争2-[125 I]碘褪黑激素与裂解或完整细胞中的人ML 1A受体结合，导致双相曲线，揭示受体的超高（约20%）和高（约80%）亲和力状态的存在。鸟苷-50-（3-硫代）三磷酸（100 μ M-30 μ M）单独加入时抑制2-[125 I]碘褪黑激素结合（IC 50 = 0.87 +/- 0.12 μ M;三个实验），表明受体与G蛋白解偶联。此外，鸟苷-5 '-O-（3-硫代）三磷酸（3 μ M）使2-[125 I]碘褪黑激素的超高和高结合褪黑激素亲和力向右偏移，导致双相曲线。褪黑激素（0.1 fM-1 nM）以浓度依赖性和双相方式抑制毛喉素诱导的cAMP形成。低浓度的褪黑激素（0.01 fM-1 PM）抑制毛喉素（100 μ M）刺激的cAMP形成，IC 50为0.1 +/- 0.05 PM（4个实验），最大抑制作用（26%）在1 PM。较高浓度的褪黑激素（1 PM-1 nM）抑制毛喉素诱导的cAMP形成，IC 50为64 +/- 1.8 PM（4次实验），最大抑制率（74%）为1 nM。Luzindole（1 μ M）是一种竞争性褪黑激素受体拮抗剂，仅在较高浓度下拮抗褪黑激素的作用（IC 50 = 1.5 +/- 0.22 nM，pKB = -7.3;三个实验）。百日咳毒素预处理完全取消褪黑激素介导的抑制毛喉素诱导的cAMP形成通过这些受体。用不同浓度的褪黑激素（0.1 μ M-1 μ M）预处理不同时间（1、6、18和24小时）不会降低2-[125 I]碘褪黑激素结合。然而，褪黑激素对2-[125 I]碘褪黑激素与用褪黑激素预处理并洗涤的细胞的结合的竞争仅针对超高亲和力位点的单个群体（IC 50 = 1.1 +/- 0.28 nM;三个实验），如双相曲线所揭示的。用褪黑激素预处理的细胞显示了对毛喉素诱导的cAMP形成的持续抑制（约20%），这种抑制不会被广泛的洗涤（长达1小时）或当在预处理期间将luzindole（1 μ M）与褪黑激素一起加入时逆转。这些结果表明，褪黑激素与人ML 1A褪黑激素受体的超高亲和力状态的紧密结合可能是体内低浓度循环激素调节下丘脑视交叉上核信号传导的机制。
The human ML1A 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 ML1A melatonin receptor stably expressed in Chinese hamster ovary cells. Association of 2-[125I]iodomelatonin binding was maximal by 1.5 hr at 37 degrees and fully dissociated on the addition of 1 microM melatonin. The binding of 2-[125I]iodomelatonin was saturable and of high affinity (KD = 74 +/- 14 PM, Bmax = 679 +/- 88 fmol/mg protein; three experiments). The pharmacological profile of various melatonin analogues revealed a profile (2-iodomelatonin > or = melatonin > N-acetyl serotonin > luzindole) characteristic of an ML1 subtype. Competition of melatonin for 2-[125I]iodomelatonin binding to the human ML1A receptor in lysed or intact cells resulted in biphasic curves revealing the existence of super high (approximately 20%) and high (approximately 80%) affinity states of the receptor. Guanosine-5'-0-(3-thio)triphosphate (100 PM-30 microM) when added alone inhibited 2-[125I]iodomelatonin binding (IC50 = 0.87 +/- 0.12 microM; three experiments), suggesting uncoupling of the receptor from G proteins. In addition, guanosine-5'-O-(3-thio)triphosphate (3 microM) 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 microM)-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 microM), a competitive melatonin receptor antagonist, antagonized the effect of melatonin at the higher concentrations only (IC50 = 1.5 +/- 0.22 nM, pKB = -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 microM) 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 (approximately 20%) of forskolin-induced cAMP formation that was not reversed by extensive washes (up to 1 hr) or when luzindole (1 microM) was added together with melatonin during pretreatment. These results suggest that tight binding of melatonin to the super high affinity state of the human ML1A melatonin receptor may be the mechanism by which low concentrations of circulating hormone in vivo regulates signaling in the suprachiasmatic nucleus of the hypothalamus.