Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

Abstract

Abstract. Peptidylglycine o~-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and midregion lyase act in sequence to perform the peptide a-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry.Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membraneassociated were all packaged into regulated secretory granules in AtT-20 cells.

Localization of Metallocarboxypeptidase D in AtT-20 Cells

“…In contrast, soluble forms of PAM are efficiently packaged into storage granules in AtT-20 cells, indicating that the lumenal domain of PAM plays a major role in sorting into the regulated pathway (32). The mechanism of the protein sorting to the regulated secretory pathway remains unclear, and two alternative models have been proposed.…”

“…The intracellular distribution of the soluble CPD was not previously examined. Interestingly, both the soluble and membrane-associated forms of PAM are packaged into mature secretory granules in AtT-20 cells (32), indicating that the lumenal domain of PAM contains sorting information. In the present study, we further characterized the intracellular distribution of CPD using immunoisolation and electron microscopic approaches.…”

Kalirin, a Multifunctional PAM COOH-terminal Domain Interactor Protein, Affects Cytoskeletal Organization and ACTH Secretion from AtT-20 Cells

“…Expression of membrane PAM raised the basal rate of ACTH secretion 3-fold (p Ͻ 0.012 compared with nontransfected) and obliterated any significant stimulation of secretion by corticotropin-releasing hormone or BaCl 2 (Fig. 8) or by phorbol esters (22). Expression of Kalirin-(447-1124), the PAM interactor domain of Kalirin, resulted in nearly a 2-fold increase in the basal rate of ACTH secretion compared with nontransfected cells (p Ͻ 0.02).…”

“…In AtT-20 lines expressing membrane PAM, secretagogue treatment failed to stimulate ACTH secretion (22). Upon expression of Kalirin, application of secretagogue resulted in a robust stimulation of ACTH secretion (Fig.…”

“…The disposition of amidated peptide products differs with cell type, with polarized secretion from neuronal processes, prolonged storage and tightly regulated release from pituitary cells, and rapid secretion from endothelial cells and glia (21)(22)(23)(24). The identification of PAM interactors whose expression is limited to a subset of the PAM-producing cells was thus anticipated.…”