ACTH INDUCTION OF CYTOCHROME P450 IN ADRENAL CELLS

所有哺乳动物物种的正常繁殖和生命维持 需要复杂的类固醇生成活动模式。一个长期目标是 这个实验室是为了在生物化学水平上了解 参与调节编码所需酶的基因的表达 类固醇激素的生物合成。具体地说，我们把注意力集中在 类固醇羟基酶(细胞色素P450)及其相关酶的研究 需要肾上腺皮质将胆固醇转化为 糖皮质激素、盐皮质激素和肾上腺雄激素(前体 性激素)。编码这些酶的基因的表达是 涉及发育、组织特异性、非cAMP的多因素 和cAMP依赖机制。在肾上腺皮质，cAMP依赖 类固醇羟基酶表达的调节是对多肽的反应 激素ACTH来自于脑下垂体前叶，是 维持最佳的类固醇合成活性，使其生物合成 必需的类固醇激素是按需获得的。在这场竞争中 继续申请，我们建议调查唯一的 与cAMP依赖的转录相关的转录因子 人45Oc21基因、牛P450c17和P45Oscc基因以及牛 肾上腺素基因。这个实验室以前的研究已经建立了 与这些基因相关的DNA元件(CRS序列)是 在彼此和其他已知阵营之间都是独一无二的 反应基因。我们建议进行纯化、克隆和鉴定 转录因子与这些CRS元件中的每一个相关的转录因子 利用缺失和定点突变技术，在细菌中表达 重组蛋白、体外转录系统和瞬时 转染研究我们将鉴定和表征DNA结合和 每个转录因子中的转录激活域。至 评估我们将利用的cAMP反应结构域(32P)-标记，多肽 定位磷酸氨基酸的作图和蛋白质测序。在我们漫长的- 学期计划，在生化水平上了解慢性作用 ACTH调节肾上腺皮质的类固醇生成能力，这些 研究将阐明cAMP依赖的蛋白激酶的作用部位， 调节这些基因中的每一个的关键共同酶。

Normal reproduction and maintenance of life in all mammalian species requires complex patterns of steroidogenic activities. A long-term goal of this laboratory is to understand at the biochemical level the mechanisms involved in regulating expression of genes encoding enzymes required for steroid hormone biosynthesis. Specifically we are focusing our attention on the steroid hydroxylases (cytochromes P450) and related enzymes in the adrenal cortex required for the conversion of cholesterol to glucocorticoids, mineralocorticoids and the adrenal androgens (precursors of sex hormones). Expression of genes encoding these enzymes is multifactorial involving developmental, tissue specific, cAMP-independent and cAMP-dependent mechanisms. In the adrenal cortex, cAMP-dependent regulation of steroid hydroxylase expression is responsive to the peptide hormone ACTH derived from the anterior pituitary, and is required for maintenance of optimal steroidogenic activity such that biosynthesis of essential steroid hormones is achieved on demand. In this competing continuation application, we propose to investigate the unique transcription factors associated with cAMP-dependent transcription of the human 45Oc2l gene, the bovine P450c17 and P45Oscc genes, and the bovine adrenodoxin gene. Previous studies in this laboratory have established that DNA-elements (CRS sequences) associated with each of these genes are unique, both with respect to one another and to other known cAMP responsive genes. We propose purification, cloning nd characterization of the transcription factors associated with each of these CRS elements. Using deletion and site-directed mutagenesis, bacterial expression of recombinant proteins, in vitro transcription systems, and transient transfection studies we will identify and characterize the DNA-binding and transcription activation domains in each of the transcription factors. To evaluate cAMP-responsive domains we will utilize (32P]-labeling, peptide mapping and protein sequencing to locate phosphoamino acids. In our long- term plan to understand at the biochemical level the chronic action of ACTH to regulate steroidogenic capacity in the adrenal cortex, these studies will elucidate sites of action of cAMP-dependent protein kinase, a key common enzyme in regulation of each of these genes.