Lovenberg T W, Erlander M G, Baron B M, Racke M, Slone A L, Siegel B W, Craft C M, Burns J E, Danielson P E, Sutcliffe J G
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2184-8. doi: 10.1073/pnas.90.6.2184.
Sequential polymerase chain reaction experiments were performed to amplify a unique sequence representing a guanine nucleotide-binding protein (G-protein)-coupled receptor from rat hypothalamic cDNA. Degenerate oligonucleotides corresponding to conserved amino acids from transmembrane domains III, V, and VI of known receptors [5-HT1A, 5-HT1C, and 5-HT2; 5-HT is serotonin (5-hydroxytryptamine)] were used as primers for the sequential reactions. The resulting product was subcloned and used to screen a rat genomic library to identify a full-length clone (MR77) containing an intronless open reading frame encoding a 366-amino acid seven-transmembrane domain protein. The human homolog was isolated, and its encoded protein had 93% overall amino acid identity with the rat sequence. Within the conserved transmembrane domains, the sequences exhibit approximately 52%, 59%, 65%, and 68% amino acid identity with the known rat 5-HT1A, rat 5-HT1B, rat 5-HT1D, and human 5-HT1E receptors, respectively. MR77 was subcloned into a eukaryotic expression vector system and expressed in CosM6 cells. Studies on broken cell preparations indicate that the expressed receptor exhibits 125I-labeled d-lysergic acid diethylamide (LSD) binding that can be displaced by serotonin but not by other biogenic amines. The specific binding is displaced by the selective 5-HT1D agonist sumatriptan but not by the mixed 5-HT1A/1D agonist 5-carboxyamidotryptamine. 125I-labeled LSD binding was competitively antagonized by the ergot alkaloids methysergide and ergotamine. HeLa cells transfected with the MR77 gene exhibited inhibition of adenylate cyclase in response to serotonin. MR77 is expressed at low levels throughout the brain, with the greatest expression in the cortex, hippocampus, and striatum. MR77 thus represents a 5-HT receptor of the 5-HT1 class, and we propose that, based on the pharmacological characterization, MR77 represents an additional 5-HT1E-like receptor.
进行了连续聚合酶链反应实验，以从大鼠下丘脑cDNA中扩增出一段代表鸟嘌呤核苷酸结合蛋白（G蛋白）偶联受体的独特序列。对应于已知受体[5-HT1A、5-HT1C和5-HT2；5-HT即血清素（5-羟色胺）]跨膜结构域III、V和VI保守氨基酸的简并寡核苷酸用作连续反应的引物。将所得产物亚克隆，并用于筛选大鼠基因组文库，以鉴定一个全长克隆（MR77），其包含一个无内含子的开放阅读框，编码一个366个氨基酸的七跨膜结构域蛋白。分离出了人类同源物，其编码的蛋白与大鼠序列的总体氨基酸同一性为93%。在保守的跨膜结构域内，该序列与已知的大鼠5-HT1A、大鼠5-HT1B、大鼠5-HT1D和人类5-HT1E受体的氨基酸同一性分别约为52%、59%、65%和68%。将MR77亚克隆到真核表达载体系统中，并在CosM6细胞中表达。对破碎细胞制剂的研究表明，所表达的受体表现出可被血清素取代但不能被其他生物胺取代的125I标记的d-麦角酸二乙胺（LSD）结合。特异性结合可被选择性5-HT1D激动剂舒马曲坦取代，但不能被5-HT1A/1D混合激动剂5-羧酰胺色胺取代。125I标记的LSD结合受到麦角生物碱美西麦角和麦角胺的竞争性拮抗。用MR77基因转染的HeLa细胞对血清素表现出腺苷酸环化酶的抑制作用。MR77在整个大脑中低水平表达，在皮质、海马体和纹状体中表达最高。因此，MR77代表5-HT1类的5-HT受体，并且我们基于药理学特征提出，MR77代表一种额外的5-HT1E样受体。
