Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and β-lipotropin

Abstract

The pituitary cell line, AtT-20, synthesizes the precursor to corticotropin (adrenocorticotropic hormone; ACTH) and β-andorphin and correctly glycosylates and cleaves it to make the mature forms of the hormones before they are secreted. This cell line was used to study the intracellular transport, packaging, and secretion of these hormones. Secretory granules from the cells were isolated by homogenization and differential centrifugation and isopycnic sedimentation on a 2H2O-Ficoll gradient to give a preparation having a specific activity of 90 μg ACTH per mg of protein, which is 30- to 90-fold greater than that of whole cells. The granules have density characteristics and a sedimentation coefficient that are appropriate for spheres of 1000 Å radius. They contain all of the fragments of the initial ACTH/endorphin precursor but almost undetectable amounts of the intact precursor. The fragments constitute about 50% of the protein in the secretory granule fraction and, from density measurements, we estimate that they are present in ≃60,000 copies per vesicle. The cell line secretory granules appear, therefore, to be similar to mature secretory granules in normal differentiated tissues. ACTH first appears in the secretory granule at 30-45 min after synthesis. Cleavage of the precursor to mature ACTH occurs at about the same time in the whole cell. Therefore, proteolysis of the prohormone to ACTH and to β-lipotropin is a metabolic event that can be correlated with the packaging of the hormone into a mature secretory granule. Cleavage of β-lipotropin to β-endorphin occurs later, probably in the secretory granule.