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

Ciccotosto GD, Schiller MR, Eipper BA, Mains RE - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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.

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Localization of PAM and ACTH after Dox induction. Immunofluorescent staining for PAM in iPAM cells treated with the  indicated doses of Dox (micrograms per milliliter) using mAb 6E6 (A); cells were photographed under identical conditions and the corresponding phase images are shown. (B) Immunofluorescent staining of Dox-treated iPAM cells (4 μg/ml Dox for 48 h) with mAb to  PAM (6E6; green) and polyclonal antibody to TGN38 (red); images were taken in black and white and colorized using Adobe Photoshop; areas of overlap appear yellow (bottom). Arrows mark tips of processes. Images in C were obtained with the Noran confocal laser  scanning microscope. Dox-treated cells were stained simultaneously for PAM (mAb 6E6; green) and ACTH (JH93; red); the combined  images showing areas of PAM and ACTH overlap are seen in yellow (C, bottom). The cell nucleus (n) and tips of cellular processes (arrow) are indicated. Bar, 10 μm.
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Figure 6: Localization of PAM and ACTH after Dox induction. Immunofluorescent staining for PAM in iPAM cells treated with the indicated doses of Dox (micrograms per milliliter) using mAb 6E6 (A); cells were photographed under identical conditions and the corresponding phase images are shown. (B) Immunofluorescent staining of Dox-treated iPAM cells (4 μg/ml Dox for 48 h) with mAb to PAM (6E6; green) and polyclonal antibody to TGN38 (red); images were taken in black and white and colorized using Adobe Photoshop; areas of overlap appear yellow (bottom). Arrows mark tips of processes. Images in C were obtained with the Noran confocal laser scanning microscope. Dox-treated cells were stained simultaneously for PAM (mAb 6E6; green) and ACTH (JH93; red); the combined images showing areas of PAM and ACTH overlap are seen in yellow (C, bottom). The cell nucleus (n) and tips of cellular processes (arrow) are indicated. Bar, 10 μm.

Mentions: To evaluate the localization of PAM at different levels of expression, a dose–response curve for Dox induction of PAM-1 in iPAM cells was examined by immunofluorescence (Fig. 6 A). Cells were fixed and visualized with a mAb directed against the PAM-1 COOH terminus. Less than 1% of the cells stained for PAM-1 when the untreated iPAM cells were examined (Fig. 6 A, 0 Dox); as for Western blots, our antisera cannot detect the endogenous levels of PAM in AtT-20 cells. In iPAM cells treated for 48 h at a low Dox dose (0.25 μg/ml), PAM-1 signal was seen in ∼50% of the cells (Fig. 6 A, 0.25 Dox). In maximally induced iPAM cells (4 μg/ml Dox), PAM staining was much more intense and >95% of the cells displayed fluorescence (Fig. 6 A, 4 Dox).


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

Ciccotosto GD, Schiller MR, Eipper BA, Mains RE - J. Cell Biol. (1999)

Localization of PAM and ACTH after Dox induction. Immunofluorescent staining for PAM in iPAM cells treated with the  indicated doses of Dox (micrograms per milliliter) using mAb 6E6 (A); cells were photographed under identical conditions and the corresponding phase images are shown. (B) Immunofluorescent staining of Dox-treated iPAM cells (4 μg/ml Dox for 48 h) with mAb to  PAM (6E6; green) and polyclonal antibody to TGN38 (red); images were taken in black and white and colorized using Adobe Photoshop; areas of overlap appear yellow (bottom). Arrows mark tips of processes. Images in C were obtained with the Noran confocal laser  scanning microscope. Dox-treated cells were stained simultaneously for PAM (mAb 6E6; green) and ACTH (JH93; red); the combined  images showing areas of PAM and ACTH overlap are seen in yellow (C, bottom). The cell nucleus (n) and tips of cellular processes (arrow) are indicated. Bar, 10 μm.
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Figure 6: Localization of PAM and ACTH after Dox induction. Immunofluorescent staining for PAM in iPAM cells treated with the indicated doses of Dox (micrograms per milliliter) using mAb 6E6 (A); cells were photographed under identical conditions and the corresponding phase images are shown. (B) Immunofluorescent staining of Dox-treated iPAM cells (4 μg/ml Dox for 48 h) with mAb to PAM (6E6; green) and polyclonal antibody to TGN38 (red); images were taken in black and white and colorized using Adobe Photoshop; areas of overlap appear yellow (bottom). Arrows mark tips of processes. Images in C were obtained with the Noran confocal laser scanning microscope. Dox-treated cells were stained simultaneously for PAM (mAb 6E6; green) and ACTH (JH93; red); the combined images showing areas of PAM and ACTH overlap are seen in yellow (C, bottom). The cell nucleus (n) and tips of cellular processes (arrow) are indicated. Bar, 10 μm.
Mentions: To evaluate the localization of PAM at different levels of expression, a dose–response curve for Dox induction of PAM-1 in iPAM cells was examined by immunofluorescence (Fig. 6 A). Cells were fixed and visualized with a mAb directed against the PAM-1 COOH terminus. Less than 1% of the cells stained for PAM-1 when the untreated iPAM cells were examined (Fig. 6 A, 0 Dox); as for Western blots, our antisera cannot detect the endogenous levels of PAM in AtT-20 cells. In iPAM cells treated for 48 h at a low Dox dose (0.25 μg/ml), PAM-1 signal was seen in ∼50% of the cells (Fig. 6 A, 0.25 Dox). In maximally induced iPAM cells (4 μg/ml Dox), PAM staining was much more intense and >95% of the cells displayed fluorescence (Fig. 6 A, 4 Dox).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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.

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