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.
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