Acute Hormonal Regulation of Steroidogenesis

描述（由申请人提供）：尚不清楚胆固醇诱导的cAMP水平在线粒体中激活胆固醇转运和类固醇合成的确切机制。我们认为这种机制涉及一种大分子信号复合物，其中一种新鉴定的外周型苯二氮卓受体（PBR）相关蛋白（PAP 7）结合cAMP依赖性蛋白激酶（PKA）的调节亚基RI-α，从而允许局部底物类固醇生成急性调节蛋白（星星）的（线粒体）有效催化活化和磷酸化，导致胆固醇通过高亲和力胆固醇结合蛋白PBR转移到线粒体内膜中。克隆了小鼠和人PAP 7蛋白，表征了它们的基因组组织、组织分布和Leydig细胞中的亚细胞定位。PAP 7在类固醇生成组织中高度表达，其中它遵循PKA-RI-α表达模式，并且来自人肾上腺疾病的数据表明它参与PKA-RI-α介导的肿瘤发生和非依赖性皮质醇增多症。PAP 7定位于高尔基体和线粒体中，并且抑制PAP 7表达导致降低的胆固醇诱导的胆固醇转运到线粒体中和降低的类固醇形成。总之，这些数据表明，PAP 7作为A-激酶锚定蛋白（AKAP），一种“支架”蛋白，靶向cAMP信号传导到线粒体，类固醇合成开始。这些研究将扩展到四个具体目标。在第一个目标中，我们将建立时间和空间的关系，存在于这个线粒体cAMP转导体的组件和激素治疗的影响。在第二个目标中，我们将研究定义复合物的各种组分之间相互作用的结合和特异性的分子决定因素。在第三个目标中，我们将研究PKA-RI-alphaPAP 7/PBR组装是否促进星星磷酸化和胆固醇转运到线粒体中。在第四个目标中，我们将通过在动物和类固醇生成细胞水平上的基因靶向来确定“分子开关”PAP 7的体内功能。从这些研究中产生的数据应该测试我们的假设，即线粒体cAMP信号传导复合物（transtranstranseosome）指导和放大cAMP的作用，导致类固醇生成的诱导和维持。

DESCRIPTION (provided by applicant): The precise mechanism by which hormone-induced cAMP levels act at mitochondria to activate cholesterol transport and steroid synthesis is unknown. We propose that this mechanism involves a macromolecular signaling complex where a newly identified peripheral-type benzodiazepine receptor (PBR)-associated protein (PAP7) binds the regulatory subunit RI-alpha of the cAMP-dependent protein kinase (PKA), thus allowing for local (mitochondrial) efficient catalytic activation and phosphorylation of the substrate steroidogenesis acute regulatory protein (StAR), leading to cholesterol transfer through the high affinity cholesterol binding protein PBR into the inner mitochondrial membrane. The mouse and human PAP7 proteins were cloned, their genomic organization, tissue distribution and subcellular localization in Leydig cells characterized. PAP7 is highly expressed in steroidogenic tissues, where it follows the pattern of PKA-RI-alpha expression and data from a human adrenal disease suggest that it participates in PKA-RI-alpha-mediated tumorigenesis and hormone-independent hypercortisolism. PAP7 is localized in the Golgi and mitochondria and inhibition of PAP7 expression results in reduced hormone-induced cholesterol transport into mitochondria and decreased steroid formation. Taken together these data suggest that PAP7 functions as an A-kinase anchoring protein (AKAP), a "scaffold" protein that targets cAMP signaling to mitochondria where steroidogenesis begins. These studies will be extended with four specific aims. In the first aim we will establish the temporal and spatial relationship of the components present in this mitochondrial cAMP transduceosome and the effects of hormone treatment. In the second aim, we will examine the molecular determinants defining the binding and specificity of the interaction among the various components of the complex. In the third aim, we will investigate whether the PKA-RI-alphaPAP7/PBR assembly facilitates StAR phosphorylation and cholesterol transport into mitochondria. In the fourth aim, we will determine the in vivo function of the "molecular switch" PAP7 by gene targeting at the animal and steroidogenic cell levels. Data generated from these studies should test our hypothesis that a mitochondrial cAMP signaling complex (transduceosome) directs and amplifies the effects of cAMP leading to the induction and maintenance of steroidogenesis.