A common precursor to ACTH and β-endorphin: hormonal control, structure, processing and purification of structural gene fragments

ACTH, β-endorphin and β-lipotropin (β-LPH) are contained in a single peptide produced by AtT-20 cells. The precursor's mRNA (ACTH/LPH mRNA) in heterologous cell-free systems (wheat germ, etc.) produces a nonglycosylated 28,000 dalton protein. The earliest detectable cellular product is a 29,000 glycoprotein with tryptic peptides identical to the cell-free product. Some product is glycosylated to species of 32,000 or 34,000. Proteolytic processing converts the 29,000 to a 21,000 and then a 4,500 form (α-(1-39)ACTH), the 32,000 to a 23,000 and then a 13,000 form (glycosylated α-(1-39)ACTH), and the 34,000 to a 26,000 and then α-(1-39)ACTH. The first proteolysis yields nonglycosylated β-endorphin-containing β-LPH. The second produces an unidentified glycosylated peptide. Glucocorticoids decrease ACTH production and ACTH/LPH mRNA, but do not affect total RNA or protein synthesis, cell growth or processing. ACTH/LPH mRNA has been further purified and double-stranded cDNA complementary to it prepared and cleaved with restriction enzymes. These yield bands highly enriched with sequences of the ACTH/LPH gene. Thus, processing of this precursor involves glycosylation and proteolysis to produce several forms of ACTH, β-endorphin-containing β-LPH and another peptide, possibly a new unidentified hormone. Unlike other secretory proteins, a signal peptide has not been detected. Glucocorticoids can specifically inhibit ACTH and β-endorphin production by decreasing their mRNA. Thus, steroids do not always stimulate transcription of specific mRNAs. Finally, purified fragments of the structural ACTH/LPH gene provide material for sequencing and cloning.