Acute Hormonal Regulation of Steroidogenesis

描述(由申请人提供)：激素诱导的cAMP水平作用于线粒体以激活胆固醇运输和类固醇合成的确切机制尚不清楚。我们认为，这种机制涉及一个大分子信号复合体，其中新发现的外周型苯二氮卓类受体(PBR)相关蛋白(PAP7)与cAMP依赖的蛋白激酶(PKA)的调节亚基RI-α结合，从而允许底物类固醇合成急性调节蛋白(STAR)的局部(线粒体)高效催化激活和磷酸化，导致胆固醇通过高亲和力胆固醇结合蛋白PBR转移到线粒体膜内。克隆了小鼠和人PAP7蛋白，并对其基因组结构、组织分布和在间质细胞中的亚细胞定位进行了研究。PAP7在类固醇生成组织中高表达，它遵循PKA-RI-α的表达模式，来自人类肾上腺疾病的数据表明，它参与了PKA-RI-α介导的肿瘤发生和激素非依赖性皮质醇增多症。PAP7定位于高尔基体和线粒体，抑制PAP7的表达会减少激素诱导的胆固醇向线粒体的转运，并减少类固醇的形成。综上所述，这些数据表明，PAP7发挥着A-激酶锚定蛋白(AKAP)的功能，AKAP是一种以cAMP信号为靶点的“支架”蛋白，线粒体是类固醇合成的起点。这些研究将扩展到四个具体目标。在第一个目标中，我们将建立线粒体cAMP转导小体中各组分的时间和空间关系以及激素处理的效果。在第二个目标中，我们将检查定义络合物不同成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-alphaPAP7/PBR组装是否促进STAR磷酸化和胆固醇向线粒体的运输。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”PAP7在体内的功能。这些研究产生的数据应该验证我们的假设，即线粒体cAMP信号复合体(转导体)指导并放大cAMP的作用，导致类固醇的诱导和维持。

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.