MOLECULAR PHARMACOLOGY OF THE PACAP RECEPTOR

该项目的长期目标是了解分子 肽的激动剂识别和G蛋白偶联机制 受体的研究，阐明肽受体的作用机制 脱敏 垂体腺苷酸环化酶激活肽 （PACAP）是最近发现的38个氨基酸的神经肽，其在神经细胞中被鉴定。 下丘脑提取物通过其能力，有力地和有力地激活 培养的垂体细胞腺苷酸环化酶活性。 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与血管活性肽高度同源， 肠肽（VIP），最重要的肽介质之一， 冠状动脉、脑动脉和外周动脉血管舒张。 VIP是一个 胰高血糖素/胰泌素家族的神经肽成员，并且通过同源性 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CNS和外周组织中。 II型 受体似乎也以高亲和力结合VIP。VIP及 分泌素受体cDNA最近已被克隆，但同源性很小 存在。 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的亚家族。 的 PACAP受体的cDNA的克隆将极大地 增强对VIP/PACAP受体亚家族的理解。 因此，本申请的具体目的包括：（1） 垂体腺苷酸cDNA的分子克隆 环化酶激活肽（PACAP）受体;（2）分子和 PACAP受体cDNA的药理学表征;和（3） 评价激动剂结合和G蛋白偶联关系， VIP和PACAP受体。 一种基于COS的表达克隆方法 细胞用于分离PACAP受体cDNA。 完成 随后将进行药理学评价，包括对 激动剂和G蛋白结合重要区域的受体。

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.