PARTIAL SEQUENCING OF THE RAT STEROIDOGENIC ACUTE REGULATORY PROTEIN MESSAGE FROM IMMORTALIZED GRANULOSA-CELLS - REGULATION BY GONADOTROPINS AND ISOPROTERENOL

Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involv ed in the transport of cholesterol to inner mitochondrial membrane dur ing stimulation of steroid hormone biosynthesis, has recently been clo ned from human adrenals and MA-10 mouse Leydig tumor cells. We examine d the regulation of StAR mRNA accumulation upon induction of steroidog enesis in immortalized rat granulosa cells. Granulosa cells were trans fected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) o r with FSH receptor (GFSHR17) or with the beta(2)-adrenergic receptor (G beta(2)AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoprote renol (10 mu M) or forskolin (50 mu M). By quantitative RT-PCR, StAR m RNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskol in. In contrast, variable amount of the message was detected in all st eroidogenic cell lines cotransfected with SV40 DNA and Ka-ras. Moreove r, an increase in the StAR mRNA expression was evident in all steroido genic cells upon stimulation with their respective agonists, concomita ntly with enhanced progesterone production. The RT-PCR product was seq uenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amin o acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is ex pressed only in the steroidogenic rat granulosa cells and can be upreg ulated by FSH, hCG, isoproterenol and forskolin in the appropriate cel l lines. In addition, we find that the rat StAR cDNA exhibit a high de gree of homology with the mouse and human sequences.