EXPRESSION CLONING OF A HIGH-AFFINITY MELATONIN RECEPTOR FROM XENOPUS DERMAL MELANOPHORES
EXPRESSION CLONING OF A HIGH-AFFINITY MELATONIN RECEPTOR FROM XENOPUS DERMAL MELANOPHORES
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DOI：
10.1073/pnas.91.13.6133
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
REPPERT, SM
中科院分区：
文献类型：
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作者：
EBISAWA, T;KARNE, S;REPPERT, SM
使用表达克隆策略，已从非洲爪蟾皮肤黑色素细胞中分离出高亲和力褪黑激素受体cDNA。在COS-7细胞中瞬时表达的cDNA导致高亲和力的2-[I-125]-碘褪黑素结合（Kd = 6.3 +/- 0.3 × 10（-11）M）。此外，6个配体表现出抑制特异性2-[I-125]碘褪黑激素结合的等级顺序，与内源性高亲和力受体的报告相同。稳定表达受体cDNA的CHO细胞的功能研究表明，褪黑激素通过克隆的受体作用抑制毛喉素刺激的cAMP积累的剂量依赖性的方式。北方印迹分析表明，褪黑激素受体转录本适度表达在非洲爪蟾皮肤黑色素细胞。该cDNA编码一个420个氨基酸的蛋白质，该蛋白质含有7个疏水片段。结构分析表明，该受体蛋白是一个新发现的鸟嘌呤核苷酸结合蛋白偶联受体家族成员。
Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[I-125]- iodomelatonin binding (K-d = 6.3 +/- 0.3 X 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[I-125] iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.