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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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Induction of PAM mRNA in iPAM cells. Northern  blot of total RNA (10 μg) isolated from iPAM cells after treatment with the indicated doses of Dox (μg/ml) for 48 h. The membrane was hybridized with cDNA probes for PHM (top) and S26  ribosomal protein (bottom). Similar results were obtained in two  other analyses of this type.
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Figure 2: Induction of PAM mRNA in iPAM cells. Northern blot of total RNA (10 μg) isolated from iPAM cells after treatment with the indicated doses of Dox (μg/ml) for 48 h. The membrane was hybridized with cDNA probes for PHM (top) and S26 ribosomal protein (bottom). Similar results were obtained in two other analyses of this type.

Mentions: Induction of PAM-1 mRNA was examined by Northern blot (Fig. 2). iPAM cells were treated with increasing concentrations of Dox for 48 h. In the exposure shown, there is no detectable PAM mRNA in the absence of Dox and the intensity of the signal increases with higher doses of Dox, reaching maximal expression levels at 1–4 μg/ml Dox. With longer exposure times, the level of PAM mRNA in the noninduced iPAM cells was found to be similar to the level of endogenous PAM mRNA in the nontransfected cells (data not shown). There was a 60-fold induction of PAM mRNA expression in the maximally induced iPAM cells compared with the noninduced cells. No decrease in PAM-1 mRNA level was observed with the highest dose of Dox (8 μg/ml). The level of PAM-1 mRNA in the maximally induced cells was similar to the level in stably transfected PAM-1 cells (data not shown).


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)

Induction of PAM mRNA in iPAM cells. Northern  blot of total RNA (10 μg) isolated from iPAM cells after treatment with the indicated doses of Dox (μg/ml) for 48 h. The membrane was hybridized with cDNA probes for PHM (top) and S26  ribosomal protein (bottom). Similar results were obtained in two  other analyses of this type.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132922&req=5

Figure 2: Induction of PAM mRNA in iPAM cells. Northern blot of total RNA (10 μg) isolated from iPAM cells after treatment with the indicated doses of Dox (μg/ml) for 48 h. The membrane was hybridized with cDNA probes for PHM (top) and S26 ribosomal protein (bottom). Similar results were obtained in two other analyses of this type.
Mentions: Induction of PAM-1 mRNA was examined by Northern blot (Fig. 2). iPAM cells were treated with increasing concentrations of Dox for 48 h. In the exposure shown, there is no detectable PAM mRNA in the absence of Dox and the intensity of the signal increases with higher doses of Dox, reaching maximal expression levels at 1–4 μg/ml Dox. With longer exposure times, the level of PAM mRNA in the noninduced iPAM cells was found to be similar to the level of endogenous PAM mRNA in the nontransfected cells (data not shown). There was a 60-fold induction of PAM mRNA expression in the maximally induced iPAM cells compared with the noninduced cells. No decrease in PAM-1 mRNA level was observed with the highest dose of Dox (8 μg/ml). The level of PAM-1 mRNA in the maximally induced cells was similar to the level in stably transfected PAM-1 cells (data not shown).

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