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释放的中央控制。来研究分子的级联 由CRF启动的事件刺激ACTH释放，我们使用了一个肿瘤 取自小鼠垂体前叶的ATT-20细胞系。这些细胞 提供了一个研究刺激-分泌耦合的模型系统 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依赖机制刺激ACTH释放 由于cAMP依赖的蛋白激酶活性被阻断，通过插入 将蛋白激酶抑制剂注入完整的ATT-20细胞，可防止 促肾上腺皮质激素释放作为对CRF的反应。CAMP依赖之间的联系 蛋白激酶和生物反应通常涉及蛋白质 磷酸化。因此，为了识别以下生化事件 CAMP依赖的蛋白激酶激活导致ACTH释放 对CRF的反应，受CRF调控的ATT-20细胞中的磷酸蛋白将 用双向凝胶电泳法和磷酸多肽进行表征 映射。CRF通过cAMP依赖蛋白调节的那些磷蛋白 机制将通过将蛋白激酶抑制剂插入到 CRF刺激前的ATT-20细胞。除了刺激ACTH 释放，CRF诱导阿片黑色素原(POMC)的合成， 促肾上腺皮质激素的前体。CRF可提高AtT-20细胞POMC基因表达水平。这 这种影响可能是由于POMC基因的激活或由于稳定了 POMC基因的表达。为了测试前一种可能性，我们将使用重组DNA 慢性肾衰对POMC基因率影响的方法学研究 POMC基因启动子对RNA的转录和亲和力 聚合酶。对于POMC基因启动子的研究，ATT-20细胞将 将含有细菌氯霉素的重组质粒转入 乙酰转移酶编码序列受POMC基因控制 推动者。如果发现CRF调节POMC基因-启动子亲和力 RNA聚合酶，然后对部分启动子进行突变 启动以确定CRF控制下的启动子区域。自.以来 CRF也通过cAMP依赖刺激POMC基因的表达 机制，我们将尝试确定ACTH的调节是否 这种多肽的释放和合成是通过相似或不同的 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.