The ultimate aim of this research is to describe the subcellular signaling pathway(s) used by trophic peptide hormones and other extracellular, physiologically-relevant factors to increase the rate of steroid hormone biosynthesis in steroidogenic tissues. The rate enhancement is produced by an increase in the rate of the initial, rate-limiting step in the pathway, the oxidative removal of the side chain from cholesterol to produce pregnenolone; this P450scc-catalyzed reaction occurs in the inner mitochondrial membrane. Stimulation of steroidogenesis requires both activation of a kinase and uninterrupted cytosolic protein synthesis. This latter observation lead to the hypothesis that the synthesis of a """"""""labile"""""""" protein was required to mediate the stimulatory response. Several proteins (peptides) have been proposed as mediators, among which is the mitochondrial phosphoprotein pp30 (formerly ib) and its precursor (pp37). When the cells are treated with stimulatory agent, the dose-dependence and kinetics of accumulation of pp30 correlate with stimulation of steroidogenesis in human, bovine, mouse and rat adrenal cortex, in rat corpus luteum and in rat and mouse Leydig cells. Additionally since pp37 is rapidly (tl/2 = 3-4 min) translocated into the mitochondrion and processed proteolytically to the smaller forms, pp37 is """"""""labile"""""""". During this grant period, amino acid sequence data, that we have obtained, will be used to clone the 37 kDa precursor. A bovine adrenal cDNA library will be screened using oligonucleotides, synthesized according to this sequence data, to identify the cDNA for p37. Clones will be verified by sequencing the DNA, its protein product and by other standard methods. The ability of this protein to stimulate steroidogenesis will be assessed by: (a) using the catalytic subunit of protein kinase A to phosphorylate the cloned protein in vitro and adding this phosphoprotein to mitochondria whose outer membrane has been loaded with cholesterol; the larger form of the protein is needed for reconstitution studies because it is this form of the protein that is translocated into the mitochondrion and processed proteolytically to become pp30 and (b) transfecting the cloned protein into COS l cells that have been transfected to express P450scc and adrenodoxin. Such cells should show an enhanced rate of steroid hormone synthesis when they are exposed to cAMP analogue. If successful, this combination of reconstitution studies carried out on isolated mitochondria in vitro and in transfected cells will allow a direct demonstration of the efficacy of pp37 in stimulating steroidogenesis. Additional experiments to demonstrate this involvement indirectly will involve trying to prevent the steroidogenic response to stimuli in bovine adrenal cells by (a) microinjecting antibodies to peptide, with sequence predicted by that of the 37 kDa protein to prevent import of pp37 into the mitochondrion or (b) adding cholesterol-linked phosphorothioate oligodeoxynucleotides antisense for pp37 to inhibit synthesis of p37. These data will complement the earlier correlative studies and determine whether 37 is essential for the stimulation of steroidogenesis.
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