CYLIC AMP REGULATION OF PROTEIN SERETION

调节蛋白质分泌是一种关键的动态平衡机制 在神经和内分泌系统中。许多偷偷摸摸的 已知通过增加细胞内环磷酸腺苷(CAMP)起作用 并激活cAMP依赖的蛋白激酶。近期 分子生物学实验表明，有多种 调节(R)和催化(C)亚基的异构体 CAMP依赖的蛋白激酶虽然与功能相关 这些亚型中有哪些是未知的。前阿片黑素皮质素(POMC)是 β-内啡肽和促肾上腺皮质激素前体 促肾上腺皮质激素(ACTH)与POMC来源的分泌调节 多肽已经得到了广泛的研究。一种垂体前叶肿瘤细胞 被命名为ATT-20的系列已经被描述为模仿 CAMP对POMC衍生肽分泌的调节作用 促肾上腺皮质激素释放因子、肾上腺素和生长抑素 在正常垂体前叶促肾上腺皮质激素细胞中。这项研究建议 描述生化、免疫学和分子生物学。 使用ATT-20细胞系的实验旨在 确定R和C亚基亚基异构体在 CAMP对POMC衍生肽分泌的调节。这个 具体目标包括：(1)R和C亚基的特性 AtT-20细胞中存在的亚型，(2)鉴定 亚基异构体参与介导cAMP反应 不同促分泌剂的活性成分鉴定 全酶复合体和过表达的mRNAs 各种亚基异构体，(3)确定结构特征 调停阵营所需的C亚基 蛋白质分泌的调节，以及(4)初步特征 在AtT-20细胞中C亚单位的底物蛋白。在 短期内，这些实验有望增加我们的 对cAMP依赖的蛋白激酶作为信号的理解 换能器系统和从长远来看所获得的知识 从这些实验中得到的信息有望有助于识别 CAMP的重要生理底物蛋白- 依赖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.