Kursar J D, Nelson D L, Wainscott D B, Cohen M L, Baez M
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285.
Mol Pharmacol. 1992 Oct;42(4):549-57.
Using the polymerase chain reaction amplification technique in conjunction with conventional cloning techniques, we have isolated a novel member of the serotonin [5-hydroxytryptamine (5-HT)] 1C/2 receptor subfamily (designated 5-HT2F) from rat stomach fundus. Two DNA fragments were amplified from cDNA synthesized from rat stomach fundus poly(A)+ RNA using the polymerase chain reaction technique with degenerate oligonucleotide primers derived from sequence comparisons of the second, third, and sixth putative transmembrane domains of known 5-HT receptors. These fragments were used as hybridization probes to isolate full length cDNA clones from rat stomach fundus cDNA libraries. Full length cDNA clones contained one open reading frame encoding a 479-amino acid protein with seven hydrophobic domains, characteristic of members of the guanine nucleotide-binding protein-coupled receptor superfamily. Within these seven putative transmembrane domains, the 5-HT2F receptor shared greatest homology with the rat 5-HT1C and 5-HT2 receptor subtypes (70% and 68%, respectively). Cell lines stably expressing the 5-HT2F receptor were established and demonstrated functional coupling to phosphatidylinositol hydrolysis upon 5-HT stimulation analagous to that observed for the 5-HT1C and 5-HT2 receptors. Membranes from the stably transfected cell lines (but not the untransfected parental lines) exhibited high affinity (Kd = 7.9 nM), saturable binding of [3H]5-HT. Maximum binding ranged from 0.1 to 2.4 pmol/mg of protein, depending on the clonal isolate. Using [3H]5-HT as the basis for a radioligand binding assay, the relative affinities of several tryptamine and piperazine derivatives for the cloned 5-HT2F receptor correlated with their relative potencies to contract the rat stomach fundus. These data suggest a probable relationship between this novel 5-HT2F receptor and the serotonin contractile receptor of the rat stomach fundus.
运用聚合酶链反应扩增技术并结合传统克隆技术，我们从大鼠胃底中分离出了血清素[5-羟色胺（5-HT）] 1C/2受体亚家族的一个新成员（命名为5-HT2F）。使用聚合酶链反应技术，以从已知5-HT受体的第二、第三和第六个假定跨膜结构域的序列比较中得出的简并寡核苷酸引物，从大鼠胃底poly(A)+ RNA合成的cDNA中扩增出两个DNA片段。这些片段用作杂交探针，从大鼠胃底cDNA文库中分离全长cDNA克隆。全长cDNA克隆包含一个开放阅读框，编码一个具有七个疏水结构域的479个氨基酸的蛋白质，这是鸟嘌呤核苷酸结合蛋白偶联受体超家族成员的特征。在这七个假定的跨膜结构域中，5-HT2F受体与大鼠5-HT1C和5-HT2受体亚型的同源性最高（分别为70%和68%）。建立了稳定表达5-HT2F受体的细胞系，并证明在5-HT刺激下其与磷脂酰肌醇水解功能偶联，类似于5-HT1C和5-HT2受体的情况。稳定转染细胞系（而非未转染的亲本系）的膜显示出对[3H]5-HT的高亲和力（Kd = 7.9 nM）、可饱和结合。最大结合量根据克隆分离株的不同，在0.1至2.4 pmol/mg蛋白质之间。以[3H]5-HT作为放射性配体结合测定的基础，几种色胺和哌嗪衍生物对克隆的5-HT2F受体的相对亲和力与其收缩大鼠胃底的相对效力相关。这些数据表明这种新的5-HT2F受体与大鼠胃底的血清素收缩受体之间可能存在关系。
