CYLIC AMP REGULATION OF PROTEIN SERETION

调节蛋白质分泌是一个关键的稳态机制 在神经和内分泌系统中。 许多促分泌素 已知通过增加细胞内环腺苷酸（cAMP） 水平和激活cAMP依赖性蛋白激酶。 最近 分子生物学实验已经显示出多种 调节（R）和催化（C）亚基的同种型， cAMP依赖性蛋白激酶虽然功能相关 这些异构体是未知的。 前阿黑皮素（POMC）是 β-内啡肽和促肾上腺皮质激素的前体 促肾上腺皮质激素（ACTH）和POMC衍生的分泌调节 肽已被充分研究。 垂体前叶肿瘤细胞 线，指定为AtT-20，已被表征为模仿 cAMP介导的POMC衍生肽分泌的调节 促肾上腺皮质激素释放因子、肾上腺素和生长抑素 正常垂体前叶促肾上腺皮质激素细胞。 本研究提案 描述了生物化学、免疫学和分子生物学 使用AtT-20细胞系的实验，其被设计为 确定R和C亚基同种型在 POMC衍生肽分泌的cAMP调节。 的 具体目标包括：（1）R和C亚基的表征 AtT-20细胞中存在的同种型，（2）鉴定 亚单位同种型参与介导cAMP应答， 各种促分泌素，通过识别激活的 全酶复合物和过表达的mRNA， 各种亚基异构体，（3）确定结构特征 C亚基的重要性， 蛋白质分泌的调节，以及（4）初始表征 AtT-20细胞中C亚基的底物蛋白。 在 短期内，这些实验有望增加我们的 cAMP依赖性蛋白激酶作为信号的理解 换能系统，从长远来看， 从这些实验中，预计将有助于识别 cAMP的重要生理底物蛋白- 依赖性蛋白激酶参与蛋白质的调节 分泌物

Regulation of protein secretion is a key homeostatic mechanism in both the nervous and endocrine systems. Many secretagogues are known to act by increasing intracellular cyclic AMP (cAMP) levels and activating a cAMP-dependent protein kinase. Recent molecular biological experiments have shown a multiplicity of isoforms for the regulatory (R) and catalytic (C) subunits for cAMP-dependent protein kinase although the functional relevance of these isoforms is unknown. Proopiomelanocortin (POMC) is the precursor to both beta-endorphin and adrenocorticotrophic hormone (ACTH) and the regulation of secretion of POMC-derived peptide has been well studied. An anterior pituitary tumor cell line, designated AtT-20, has been characterized that mimics the cAMP-mediated regulation of POMC-derived peptide secretion by corticotropin releasing factor, epinephrine, and somatostatin seen in normal anterior pituitary corticotrophs. This research proposal describes biochemical, immunological, and molecular biological experiments using the AtT-20 cell line which are designed to determine the functional roles of the R and C subunit isoforms in the cAMP regulation of POMC-derived peptide secretion. The specific aims include: (1) characterization of the R and C subunit isoforms present in AtT-20 cells, (2) identification of which subunit isoforms are involved in mediating the cAMP response to various secretagogues by identifying the components of activated holoenzyme complexes and by overexpressing mRNAs for the various subunit isoforms, (3) determining the structural features of the C subunits that are necessary for mediating the cAMP regulation of protein secretion, and (4) an initial characterization of substrate proteins for the C subunit in AtT-20 cells. In the short term, these experiments are expected to increase our understanding of the cAMP-dependent protein kinase as a signal transducing system and in the long term the knowledge derived from these experiments are expected to facilitate identification of important physiological substrate proteins for the cAMP- dependent protein kinase involved in the regulation of protein secretion.