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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 in stable  cell lines. Immunofluorescent  staining of nontransfected,  stably transfected PAM-1  and PHM4 cells for PAM and  ACTH (Ab JH93) (6E6 and  18E5, respectively). PAM  staining was detected with  goat anti–mouse FITC and  ACTH staining was visualized with goat anti–rabbit  Cy3. Phase-contrast images  are also shown. All cells were  photographed under identical conditions. The cell nucleus (n) and tips of cellular  processes (arrows) are indicated. Bar, 10 μm.
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Figure 7: Localization of PAM and ACTH in stable cell lines. Immunofluorescent staining of nontransfected, stably transfected PAM-1 and PHM4 cells for PAM and ACTH (Ab JH93) (6E6 and 18E5, respectively). PAM staining was detected with goat anti–mouse FITC and ACTH staining was visualized with goat anti–rabbit Cy3. Phase-contrast images are also shown. All cells were photographed under identical conditions. The cell nucleus (n) and tips of cellular processes (arrows) are indicated. Bar, 10 μm.

Mentions: Having established that the iPAM cells can be used effectively to control the expression of PAM in a given cell population, the effects of increased PAM expression on ACTH localization were evaluated by immunofluorescence (Figs. 6 C and 7). Fig. 7 shows epifluorescence images of populations of nontransfected and PAM-1–expressing cells whereas Fig. 6 C shows confocal images of two cells among a population of iPAM cells treated with Dox; as occasionally happens, only one of the pair of cells is expressing high levels of PAM (green). In the population of nontransfected (NT) cells (Fig. 7, left), vesicular, punctate staining for ACTH is observed throughout the cytosol with increased staining in the cellular processes and a slight increase in ACTH staining in the TGN region. In the single iPAM cell not expressing high levels of PAM (Fig. 6 C, left cell), the ACTH (red) staining pattern is similar to that of the nontransfected cells. pUHD cells treated with Dox also displayed ACTH staining patterns similar to those seen for nontransfected cells (data not shown). Interestingly, in the confocal image of the iPAM cell expressing PAM-1 (Fig. 6 C, right), the ACTH distribution is dramatically altered, with marked localization of ACTH to the TGN region of the cell and less intense staining of processes. Similar patterns of ACTH staining are observed in the population of AtT-20 cells expressing PAM-1 (Fig. 7, middle). The PAM and ACTH staining patterns of the Dox-treated iPAM cells (Fig. 6 C, right cell) are almost identical to those of the stably transfected PAM-1 cells (Fig. 7, PAM-1).


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 in stable  cell lines. Immunofluorescent  staining of nontransfected,  stably transfected PAM-1  and PHM4 cells for PAM and  ACTH (Ab JH93) (6E6 and  18E5, respectively). PAM  staining was detected with  goat anti–mouse FITC and  ACTH staining was visualized with goat anti–rabbit  Cy3. Phase-contrast images  are also shown. All cells were  photographed under identical conditions. The cell nucleus (n) and tips of cellular  processes (arrows) are indicated. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 7: Localization of PAM and ACTH in stable cell lines. Immunofluorescent staining of nontransfected, stably transfected PAM-1 and PHM4 cells for PAM and ACTH (Ab JH93) (6E6 and 18E5, respectively). PAM staining was detected with goat anti–mouse FITC and ACTH staining was visualized with goat anti–rabbit Cy3. Phase-contrast images are also shown. All cells were photographed under identical conditions. The cell nucleus (n) and tips of cellular processes (arrows) are indicated. Bar, 10 μm.
Mentions: Having established that the iPAM cells can be used effectively to control the expression of PAM in a given cell population, the effects of increased PAM expression on ACTH localization were evaluated by immunofluorescence (Figs. 6 C and 7). Fig. 7 shows epifluorescence images of populations of nontransfected and PAM-1–expressing cells whereas Fig. 6 C shows confocal images of two cells among a population of iPAM cells treated with Dox; as occasionally happens, only one of the pair of cells is expressing high levels of PAM (green). In the population of nontransfected (NT) cells (Fig. 7, left), vesicular, punctate staining for ACTH is observed throughout the cytosol with increased staining in the cellular processes and a slight increase in ACTH staining in the TGN region. In the single iPAM cell not expressing high levels of PAM (Fig. 6 C, left cell), the ACTH (red) staining pattern is similar to that of the nontransfected cells. pUHD cells treated with Dox also displayed ACTH staining patterns similar to those seen for nontransfected cells (data not shown). Interestingly, in the confocal image of the iPAM cell expressing PAM-1 (Fig. 6 C, right), the ACTH distribution is dramatically altered, with marked localization of ACTH to the TGN region of the cell and less intense staining of processes. Similar patterns of ACTH staining are observed in the population of AtT-20 cells expressing PAM-1 (Fig. 7, middle). The PAM and ACTH staining patterns of the Dox-treated iPAM cells (Fig. 6 C, right cell) are almost identical to those of the stably transfected PAM-1 cells (Fig. 7, PAM-1).

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.

Show MeSH