Abstract
We studied the effect of the adrenocorticotropin hormone (ACTH) on the expression of the steroidogenic acute regulatory protein (StAR) in vivo in rat and hamster adrenals and also in transfection experiments using COSβ1 cells. In vivo, ACTH increased the level of StAR mRNA within 30β60 minutes and also increased the quantity of StAR, but with a 2β3βhour delay. ACTH induced the formation of many acidic StAR species as analyzed by twoβdimensional gel electrophoresis and immunoblotting. In the transfection experiments, (Bu)~2~βcAMP also induced the formation of many acidic species for the hamster StAR; in COSβ1 cells, StAR is phosphorylated mainly on serine (S) residue(s). When alanine (A) was substituted for serine, S13A, S185A, and S194A mutants had decreased StAR activity compared to wildtype, thus determining the importance of these amino acid residues in StAR action. The fullβlength WT, N46βtruncated StAR lacking its mitochondrial import sequence, and N46βS194A had similar activities, whereas N46βS185A had completely lost its activity. Our results suggest that S194, but not S185, functions in association with the mitochondrial import sequence for the initiation of StAR activation. Further studies showed that S185 is implicated in salt bridge stability, not in StAR phosphorylation, suggesting its importance for StAR folding. Thermodynamic calculations of the hamster StAR homology model based on MLN64 show that StAR can partially unfold to bind cholesterol and serve as a rapid transfer mechanism for eventual translocation into mitochondria. This is supportive of a StAR functioning either outside the mitochondria or in the mitochondrial intermembrane space. Microsc. Res. Tech. 61:288β299, 2003. Β© 2003 WileyβLiss, Inc.