MOLECULAR CHARACTERIZATION--HISTAMINE RECEPTOR SUBTYPES

长期目标是克隆新的人组胺受体 亚型，以帮助开发新的组胺H3受体 用于治疗CNS疾病的激动剂和拮抗剂。具体目标是 （1）利用聚合酶链反应（PCR）， 简并引物，以扩增由人H3受体制备的cDNA 表达组织（例如，尾状体）或细胞（例如，U373 MG星形细胞瘤），至 克隆H3受体cDNA;（2）表达筛选GPCR富集的cDNA 来源于表达人H3受体的组织或细胞的文库， 使用选择性高亲和力H3受体拮抗剂（例如，125 I-IPP或 125 I-GT-2232）;（3）产生表达人H3的稳定细胞系 受体克隆(4)利用这个H3受体克隆来寻找 通过简并引物PCR或低严格性 杂交方法前两个目标使用两个经过验证的修改 分离表达人组胺H3的cDNA克隆的策略 受体的 随后的目标是利用这个克隆的 在H3受体拮抗剂的进一步开发中， 激动剂这笔赠款将有助于开发新的治疗方法， 许多使人衰弱的CNS疾病（即，睡眠障碍， 焦虑症、阿尔茨海默病和注意力缺陷多动障碍。 拟定商业应用： 研究将朝着分离和表征cDNA的方向进行 编码H3受体的克隆，通过简并引物PCR和表达 筛选

The long-term objectives are to clone novel human histamine receptor subtypes, to aid in the development of novel histamine H3 receptor agonists and antagonists to treat CNS diseases. The Specific Aims of this grant are to: (1) utilize the polymerase chain reaction (PCR) with degenerate primers, to amplify cDNAs made from human H3 receptor expressing tissues (e.g., caudate) or cells (e.g., U373MG astrocytoma), to clone the H3 receptor cDNA; (2) expression screen a GPCR-enriched cDNA library derived from human H3 receptor expressing tissues or cells, by using a selective high affinity H3 receptor antagonist (e.g., 125I-IPP or 125I-GT-2232); (3) create a stable cell line expressing the human H3 receptor clone. (4) utilize this H3 receptor clone to search for additional subtypes via degenerate primer PCR or low stringency hybridization. The first two aims use modifications of two proven strategies to isolate cDNA clones expressing the human histamine H3 receptor. The subsequent aims are designed to utilize this cloned receptor in the further development of H3 receptor antagonists and agonists. This grant would assist in development of novel therapeutic approaches to many debilitating CNS disorders (i.e., sleep disorders, anxiety, Alzheimer's disease and attention deficit hyperactive disorder. PROPOSED COMMERCIAL APPLICATION: Research will be conducted towards isolating and characterizing a cDNA clone encoding the H3 receptor, by degenerate primer PCR and expression screening.