O'Carroll A M, Lolait S J, König M, Mahan L C
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1992 Dec;42(6):939-46.
Using the polymerase chain reaction technique with degenerative primers, we obtained from a rat pituitary cDNA library a cDNA fragment, rAP236, that exhibited considerable homology to known receptors that belong to the guanine nucleotide-binding protein (G protein)-coupled receptor superfamily. Oligonucleotides to this fragment were used as probes to obtain a full-length cDNA from the rat pituitary cDNA library. This clone, rAP6-26, encoded a 383-amino acid protein with seven putative transmembrane domains that are characteristic of G protein-coupled receptors. The predicted amino acid sequence of the rAP6-26 cDNA exhibits 56-66% homology to recently cloned somatostatin (SRIF) receptors. Membranes prepared from COS-7 cells transfected with the rAP6-26 cDNA showed specific binding of 125I-Tyr11-SRIF, thus identifying the cDNA clone as a novel SRIF receptor. Radioligand binding competition analysis using somatostatin-28 (SRIF-28) and a number of cyclic SRIF analogs revealed that SRIF-28 was the most potent competitor of 125I-Tyr11-SRIF binding, with a approximately 30-fold greater affinity for the receptor than that of SRIF. In addition, binding of 125I-Tyr11-SRIF was markedly reduced in the presence of Na+ ions and GTP, indicating coupling of rAP6-26 receptors to inhibitory G proteins in COS-7 membranes. In adenylyl cyclase assays, forskolin-induced cAMP accumulation was inhibited by SRIF and SRIF-28, thus confirming that the rAP6-26 cDNA encodes a functional receptor protein. By Northern blot analysis, a approximately 2.6 kilobase mRNA encoding the receptor was present in the pituitary but not in the liver, small intestine, kidney, pancreas, cerebellum, or cortex. Lack of receptor mRNA expression in the brain was confirmed by in situ hybridization histochemical studies. Thus, we report the cloning of a novel rat pituitary SRIF receptor, termed SSTR4, that has marked preferential affinity for SRIF-28 and is linked to inhibition of adenylyl cyclase.
利用聚合酶链反应技术和简并引物，我们从大鼠垂体cDNA文库中获得了一个cDNA片段rAP236，它与属于鸟嘌呤核苷酸结合蛋白（G蛋白）偶联受体超家族的已知受体具有相当高的同源性。用针对该片段的寡核苷酸作为探针，从大鼠垂体cDNA文库中获得了一个全长cDNA。这个克隆体rAP6 - 26编码一个含有383个氨基酸的蛋白质，具有七个推定的跨膜结构域，这是G蛋白偶联受体的特征。rAP6 - 26 cDNA的预测氨基酸序列与最近克隆的生长抑素（SRIF）受体具有56% - 66%的同源性。用rAP6 - 26 cDNA转染的COS - 7细胞制备的细胞膜显示出对125I - Tyr11 - SRIF的特异性结合，从而将该cDNA克隆鉴定为一种新型的SRIF受体。使用生长抑素 - 28（SRIF - 28）和一些环状SRIF类似物进行的放射性配体结合竞争分析表明，SRIF - 28是125I - Tyr11 - SRIF结合的最有效竞争者，其对该受体的亲和力比SRIF大约高30倍。此外，在存在Na +离子和GTP的情况下，125I - Tyr11 - SRIF的结合明显减少，表明rAP6 - 26受体与COS - 7细胞膜中的抑制性G蛋白偶联。在腺苷酸环化酶测定中，福斯可林诱导的cAMP积累受到SRIF和SRIF - 28的抑制，从而证实rAP6 - 26 cDNA编码一种功能性受体蛋白。通过Northern印迹分析，编码该受体的约2.6千碱基mRNA存在于垂体中，但不存在于肝脏、小肠、肾脏、胰腺、小脑或皮质中。原位杂交组织化学研究证实了脑中缺乏受体mRNA表达。因此，我们报道了一种新型大鼠垂体SRIF受体的克隆，命名为SSTR4，它对SRIF - 28具有明显的优先亲和力，并与腺苷酸环化酶的抑制相关联。
