Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.
ACTH; Actin; Actin genes; Actins/metabolism; Adrenocorticotropic Hormone/secretion; Animals; Aspartic acid; Aspartic Acid Endopeptidases/metabolism; Aspartic acid—Metabolism; Biological models; Biological transport; Active; Cell lines; Cytoskeleton; Cytoskeleton/metabolism; Dose-Response Relationship; Drug; Doxycycline/administration & dosage; Doxycycline/pharmacology; Drugs--Dose-response relationship; Enzyme induction; Enzyme Induction/drug effects; Gene expression; Gene Expression/drug effects; Mice; Mixed Function Oxygenases/biosynthesis; Mixed Function Oxygenases/genetics; Models; Biological; Multienzyme complexes; Proopiomelanocortin; Pro-Opiomelanocortin/metabolism; Proprotein Convertase 1; Proprotein convertases; RNA; RNA; Messenger/genetics; RNA; Messenger/metabolism
Anesthesiology | Neuroscience and Neurobiology | Pathology | Physiology
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Ciccotosto, G. D.,
Schiller, M. R.,
Eipper, B. A.,
Mains, R. E.
Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.
The Journal of Cell Biology, 144(3),