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© The Rockefeller University Press, 0021-9525/1999//459 $5.00
The Journal of Cell Biology, Volume 144, Number 3, , 1999 459-471

Induction of Integral Membrane PAM Expression in AtT-20 Cells Alters the Storage and Trafficking of POMC and PC1

Giuseppe D. Ciccotosto^*, Martin R. Schiller, Betty A. Eipper^*, and Richard E. Mains^*

^* Departments of Neuroscience and Physiology and Departments of Pathology and Anesthesiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Peptidylglycine -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.

Key Words: rTet • regulated • sorting • granules • actin

Abbreviations used in this paper: CSFM, complete serum-free medium; Dox, doxycycline; iPAM, inducible PAM; ISG, immature secretory granule; LDCV, large dense core vesicle; NT, nontransfected; PAL, peptidyl--hydroxylglycine -amidating lyase; PAM, peptidylglycine -amidating monooxygenase; PC1, prohormone convertase 1; PHM, peptidylglycine -hydroxylating monooxygenase; POMC, proopiomelanocortin; rTet, reverse tetracycline repressor system; TGN, trans-Golgi network.

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