Biosynthesis and secretion of pituitary hormones: dynamics and regulation.

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to <pH 6.0, which is both necessary and sufficient to trigger SPC3-mediated proteolytic conversion of proopiomelanocortin to ACTH. Investigation of the pH regulatory mechanism indicates that the trans-Golgi and secretory granules maintain different pH values by distinct sorting of key membrane transporters. Mathematical modeling of our data suggests that the decreasing pH values of organelles of the regulated secretory pathway is established by gradually increasing the density of active H+ pumps from the ER to Golgi while concomitantly decreasing the H+ permeability from ER to Golgi to secretory granules. An in vitro assay was developed to study the formation of processing-competent secretory granules from their processing-incompetent precursor trans-Golgi compartment. Our data suggest that ARF1-mediated sorting of proton pumps and leaks during early stages of granule formation confers processing competency to the resulting organelle. Once formed, these young granules continue to undergo membrane remodeling which results in dynamic changes in their exocytotic behavior. Two SNAREs, VAMP4 and synaptotagmin IV, enter newly formed granules but are removed from the maturing granule membrane by vesicle budding. Sorting of these proteins is correlated with the acquisition of Ca2+-triggered exocytosis and a decrease in unregulated exocytotic rate. Thus, biosynthesis and secretion of pituitary hormones are dynamically regulated by intracellular sorting events that govern the functions of their secretory carriers.