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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 filamentous actin  after Dox induction. Staining  of noninduced iPAM cells  (iPAM−) and Dox-treated  iPAM cells (iPAM+; 4 μg/ml  Dox, 48 h) using a mAb to  PAM (6E6; red) and FITC-phalloidin (green). Images  were obtained with the Bio-Rad confocal laser scanning  microscope. Bar, 25 μm.
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Figure 10: Localization of PAM and filamentous actin after Dox induction. Staining of noninduced iPAM cells (iPAM−) and Dox-treated iPAM cells (iPAM+; 4 μg/ml Dox, 48 h) using a mAb to PAM (6E6; red) and FITC-phalloidin (green). Images were obtained with the Bio-Rad confocal laser scanning microscope. Bar, 25 μm.

Mentions: Cytoskeletal organization is a powerful factor in regulated exocytosis (Muallem et al., 1995; Carbajal and Vitale, 1997). Since PAM-1 is known to interact with proteins that can indirectly affect cytoskeletal organization (Alam et al., 1997), the distribution of filamentous actin in noninduced and induced iPAM cells was examined using the mushroom toxin, phalloidin (Fig. 10). In noninduced iPAM cells expressing only their endogenous PAM protein, PAM staining was not visible (Fig. 10, iPAM−, red). Filamentous actin was found in clusters broadly distributed throughout the cell and was collected in several subplasma membrane foci (Fig. 10, iPAM−, green); although the edges of the noninduced cells were easily visible, filamentous actin was not enriched in the subplasma membrane region. As expected, after induction with Dox, PAM-1 was largely localized to the TGN region of the cell (Fig. 10, iPAM+, red). A dramatic change in the distribution of filamentous actin accompanied expression of PAM-1 (Fig. 10, iPAM+, green); filamentous actin was concentrated at the plasma membrane with numerous foci of more intense staining. The broadly distributed diffuse patches of filamentous actin were absent from the iPAM+ cells. Patches of filamentous actin were also concentrated in the TGN region of the iPAM+ cells.


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 filamentous actin  after Dox induction. Staining  of noninduced iPAM cells  (iPAM−) and Dox-treated  iPAM cells (iPAM+; 4 μg/ml  Dox, 48 h) using a mAb to  PAM (6E6; red) and FITC-phalloidin (green). Images  were obtained with the Bio-Rad confocal laser scanning  microscope. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132922&req=5

Figure 10: Localization of PAM and filamentous actin after Dox induction. Staining of noninduced iPAM cells (iPAM−) and Dox-treated iPAM cells (iPAM+; 4 μg/ml Dox, 48 h) using a mAb to PAM (6E6; red) and FITC-phalloidin (green). Images were obtained with the Bio-Rad confocal laser scanning microscope. Bar, 25 μm.
Mentions: Cytoskeletal organization is a powerful factor in regulated exocytosis (Muallem et al., 1995; Carbajal and Vitale, 1997). Since PAM-1 is known to interact with proteins that can indirectly affect cytoskeletal organization (Alam et al., 1997), the distribution of filamentous actin in noninduced and induced iPAM cells was examined using the mushroom toxin, phalloidin (Fig. 10). In noninduced iPAM cells expressing only their endogenous PAM protein, PAM staining was not visible (Fig. 10, iPAM−, red). Filamentous actin was found in clusters broadly distributed throughout the cell and was collected in several subplasma membrane foci (Fig. 10, iPAM−, green); although the edges of the noninduced cells were easily visible, filamentous actin was not enriched in the subplasma membrane region. As expected, after induction with Dox, PAM-1 was largely localized to the TGN region of the cell (Fig. 10, iPAM+, red). A dramatic change in the distribution of filamentous actin accompanied expression of PAM-1 (Fig. 10, iPAM+, green); filamentous actin was concentrated at the plasma membrane with numerous foci of more intense staining. The broadly distributed diffuse patches of filamentous actin were absent from the iPAM+ cells. Patches of filamentous actin were also concentrated in the TGN region of the iPAM+ cells.

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