MOLECULAR PHARMACOLOGY OF THE PACAP RECEPTOR

这个项目的长期目标是理解分子 多肽的激动剂识别和G蛋白偶联机制 受体，阐明多肽受体的作用机制 脱敏。垂体腺苷环化酶激活肽 PACAP是新近发现的一种由38个氨基酸组成的神经肽。 下丘脑提取物通过其强大的激活能力 培养的垂体细胞的腺苷环化酶活性。PACAP也存在 作为1-27个氨基酸的裂解产物(PACAP-27)。氨基酸序列 分析表明，PACAP-27与血管活性高度同源 肠肽(VIP)是血管紧张素转换酶活性最重要的多肽介质之一 冠状动脉、大脑和外周动脉血管扩张。VIP是一种 胰升糖素/促胰液素神经肽家族的成员 PACAP加入了这个大家庭。两种PACAP受体，分类为I型和 II，分别存在于中枢神经系统和周围组织。第II类 受体似乎也以高亲和力与VIP结合。贵宾和 分泌素受体cDNAs最近被克隆，但几乎没有同源性 是存在的。这些数据和其他数据表明，PACAP和VIP 受体可能是G蛋白偶联受体的一个新亚家族。这个 PACAP受体基因的克隆将在很大程度上 提高对VIP/PACAP受体亚家族的认识。 因此，本申请的具体目的包括：(1) 脑垂体腺苷酸编码基因的分子克隆 环化酶激活肽(PACAP)受体； PACAP受体基因的药理特性；和(3) 激动剂结合与G蛋白偶联关系的评价 VIP和PACAP受体。一种基于COS的基因克隆方法 细胞将被用来分离PACAP受体基因。完成 随后将进行药理学评估，包括对 激动剂和G蛋白结合的重要区域的受体。

The longterm aims of this project are to understand the molecular mechanisms of agonist recognition and G protein coupling of peptide receptors, and to elucidate the mechanisms of peptide receptor desensitization. The pituitary adenylate cyclase activating peptide (PACAP) is a recently discovered 38 amino acid neuropeptide identified in hypothalamic extracts by its ability to potently and powerfully activate adenylate cyclase activity in cultured pituitary cells. PACAP also exist as a 1-27 amino acid cleavage product (PACAP-27). Amino acid sequence analysis indicates that PACAP-27 is highly homologous to vasoactive intestinal peptide (VIP), one of the most important peptide mediators of coronary, cerebral, and peripheral arterial vasodilation. VIP is a member of the glucagon/secretin family of neuropeptides, and by homology PACAP joins this family. Two PACAP receptors, classified as Types I and II, are present in CNS and peripheral tissues, respectively. Type II receptors also appear to bind to VIP with high affinity. The VIP and secretin receptor cDNA's have recently been cloned, but little homology is present. These data and others suggest that the PACAP and VIP receptors may be a new sub-family of G protein coupled receptors. The cloning of the cDNA of the PACAP receptor or receptors will greatly enhance the understandings of the VIP/PACAP receptor subfamily. Therefore, the specific aims of the present application include: (1) molecular cloning of the cDNA which encodes the pituitary adenylate cyclase activating peptide (PACAP) receptor; (2) molecular and pharmacologic characterization of the PACAP receptor cDNA; and (3) evaluation of the agonist binding and G protein coupling relationships of the VIP and PACAP receptors. An expression cloning approach with COS cells will be used to isolate the PACAP receptor cDNA. Complete pharmacologic evaluation will follow, including an evaluation of the receptor for regions important for agonist and G protein binding.