Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein

Abstract

Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced in response to acute stimulation by adrenocorticotropic hormone (ACTH) or dibutyryl cAMP. The molecular weight of this protein is 28,000 (sodium dodecyl sulfate electrophoresis), and its isoelectric point is 6.5 (isoelectric focusing). Mapping of proteolytic peptides suggests that this induced protein (i) is quite similar in primary structure to another protein (p), which is produced only in nonstimulated adrenal cells. The time course of formation of protein i and its ACTH dose response closely parallel the increase of corticosteroid production in stimulated cells. The possibility that protein i is produced in response to increased levels of some steroid of the glucocorticoid pathway is precluded by the observation that inhibition of corticosteroid synthesis by aminoglutethimide does not alter the rate of production of i. Addition of cycloheximide before ACTH, which prevents stimulation of corticosteroidogenesis, also prevents formation of protein i implying that the production of protein i depends on continuing protein synthesis. [35S/32S]Methionine pulse-chase experiments, i.e. addition of excess [32S] methionine and ACTH after prelabeling with [35S]methionine, show that protein i is not produced from pre-existing protein p or other pre-existing proteins even if protein synthesis (and increased steroid production) is not inhibited. These findings exclude post-translational modification as a mechanism for the production of i but are consistent with p and i being related by cotranslational modification. Addition of cycloheximide after stimulation causes the formation of protein i to cease, but the amount of the protein does not decrease with the same kinetics as the return of corticosteroid production to its unstimulated level. [35S/32S]Methionine pulse-chase experiments imply protein i, even under conditions of ongoing ACTH stimulation and protein synthesis, is degraded with approximately the same kinetics as after cycloheximide inhibition. The close concurrence under a wide variety of experimental conditions between the appearance of protein i and the increase in adrenal corticosteroid production, coupled with the fact that the former does not occur as a result of the latter, make protein i a likely candidate for the postulated corticosteroidogenic stimulatory protein (Ferguson, J.J. (1962) Biochim. Biophys. Acta 57, 616-617). The fact that stimulation of steroidogenesis may occur via co-translational modification of a regulatory protein is an intriguing possibility which readily explains both the observed rapid and protein synthesis-dependent stimulation and the lack of dependence of stimulation on transcription (Schulster, D. (1974) Mol. Cell. Endocr. 1, 55-64).