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性模式。的长期目标 这个实验室是为了在生化水平上了解 参与调节基因编码的酶的表达， 类固醇激素生物合成。具体来说，我们正在集中注意力 类固醇羟化酶（细胞色素P450）和相关酶在 肾上腺皮质所需的胆固醇转化为 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、cAMP非依赖性 和cAMP依赖性机制。 在肾上腺皮质，cAMP依赖性 类固醇羟化酶表达的调节响应于肽 促肾上腺皮质激素促肾上腺皮质激素，来源于垂体前叶， 维持最佳的类固醇生成活性， 必需的类固醇激素是按需获得的。 在这场竞争中， 继续申请，我们建议调查独特的 与cAMP依赖性转录相关的转录因子 人45 Oc 2l基因、牛P450 c17和P45 Oscc基因以及牛 adrenodoxin基因 该实验室以前的研究已经建立了 与这些基因中的每一个相关的DNA元件（CRS序列）是 独特的，相对于彼此和其他已知的cAMP 反应基因我们建议纯化，克隆和表征 与这些CRS元件中的每一个相关的转录因子。 使用缺失和定点突变，细菌表达 重组蛋白，体外转录系统，和瞬时 转染研究，我们将确定和表征DNA结合， 每个转录因子中的转录激活结构域。到 评估cAMP反应域，我们将利用（32 P]-标记，肽 作图和蛋白质测序来定位磷酸氨基酸。 在我们漫长的- 长期计划，以了解在生化水平的慢性作用， ACTH调节肾上腺皮质类固醇生成能力，这些 研究将阐明cAMP依赖性蛋白激酶的作用位点， 一种调节这些基因的关键酶。

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.