Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1

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 (PBL) domain and a transmembrane domain. The cyt osolic tail of PAM interacts with proteins that can affect cytoskeletal org anization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PA M-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 exp ression 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 decr eased ACTH and PHM secretion in response to secretagogue stimulation, and d ecreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using t he inducible cell line model, we show that expression of integral membrane PAM: alters the organization of the actin cytoskeleton. Altered cytoskeleta l organization may then influence the trafficking and cleavage of lumenal p roteins and eliminate the ability of AtT-20 cells to secrete ACTH in respon se to a secretagogue.