CENTRAL REGULATION OF POMC GENE EXPRESSION

下丘脑肽促肾上腺皮质激素释放因子（CRF）介导 ACTH释放的中枢控制。 为了检测分子的级联反应 事件引发的CRF刺激ACTH释放，我们已经使用了肿瘤 细胞系（AtT-20）来源于小鼠垂体前叶。 这些细胞 提供了一个模型系统，用于研究刺激-分泌偶联， 促肾上腺皮质激素细胞，因为它们是分泌ACTH的同质群体， 细胞 CRF通过cAMP依赖性机制刺激ACTH释放 由于cAMP依赖性蛋白激酶活性的阻断， 蛋白激酶抑制剂进入完整的AtT-20细胞，阻止了 促肾上腺皮质激素的释放是对CRF的反应。 cAMP依赖性 蛋白激酶和生物反应通常涉及蛋白质 磷酸化 因此，为了确定以下生化事件 cAMP依赖性蛋白激酶激活导致ACTH释放 在AtT-20细胞中，CRF调节的磷酸化蛋白将对CRF产生反应， 通过双向凝胶电泳和磷酸肽 映射. CRF通过cAMP依赖性磷酸化酶调节这些磷蛋白， 将通过将蛋白激酶抑制剂插入 AtT-20细胞。 除了刺激ACTH 释放，CRF诱导阿黑皮素原（POMC）的合成， ACTH的前体。 CRF可提高AtT-20细胞POMC mRNA水平。 这 这种作用可能是由于POMC基因的激活或POMC基因的稳定化。 POMC mRNA。 为了测试前一种可能性，我们将使用重组DNA 方法检测CRF对POMC基因频率的影响 转录和POMC基因启动子对RNA的亲和力 聚合酶。 对于POMC基因启动子研究，AtT-20细胞将被 用重组质粒转染，其中细菌氯霉素 乙酰转移酶编码序列受POMC基因控制 启动子 如果发现CRF调节POMC基因-启动子亲和力， RNA聚合酶，则启动子部分的诱变将被 启动以确定CRF控制下的启动子区域。 以来 CRF还通过cAMP依赖性的转录因子激活POMC基因的表达。 机制，我们将试图确定是否调节ACTH 这种肽的释放和合成是通过类似或不同的 磷酸化事件。

The hypothalamic peptide corticotropin-releasing factor (CRF) mediates the central control of ACTH release. To examine the cascade of molecular events initiated by CRF to stimulate ACTH release, we have used a tumor cell line (AtT-20) derived from the mouse anterior pituitary. These cells provide a model system for investigating stimulus-secretion coupling in corticotrophs as they are a homogeneous population of ACTH-secreting cells. CRF stimulates ACTH release through a cAMP-dependent mechanism since blockade of cAMP-dependent protein kinase activity, by the insertion of an inhibitor of protein kinase into intact AtT-20 cells, prevents the release of ACTH in response to CRF. The link between cAMP-dependent protein kinase and biological responses usually involves protein phosphorylation. Therefore, to identify the biochemical events following cAMP-dependent protein kinase activation that lead to the ACTH release response to CRF, the phosphoproteins regulated by CRF in AtT-20 cells will be characterized by two-dimensional gel electrophoresis and phosphopeptide mapping. Those phosphoproteins regulated by CRF through a cAMP-dependent mechanism will be identified by inserting the protein kinase inhibitor into AtT-20 cells prior to CRF stimulation. In addition to stimulating ACTH release, CRF induces the synthesis of pro-opiomelanocortin (POMC), the precursor of ACTH. CRF raises POMC mRNA levels in AtT-20 cells. This effect could be due to activation of the POMC gene or to a stabilization of POMC mRNA. To test the former possibility, we will use recombinant DNA methodologies to examine the effect of CRF on the rate of POMC gene transcription and the affinity of the POMC gene promoter for RNA polymerase. For the POMC gene promoter studies, AtT-20 cells will be transfected with a recombinant plasmid in which bacterial chloramphenicol acetyltransferase coding sequences are under the control of the POMC gene promoter. If CRF is found to regulate the POMC gene-promoter affinity for RNA polymerase, then mutagenesis of portions of the promoter will be initiated to determine the region of the promoter under CRF control. Since CRF also stimulates POMC gene expression through a cAMP-dependent mechanism, we will attempt to determine whether the regulation of ACTH release and synthesis by this peptide is through similar or distinct phosphorylation events.