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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 protein in iPAM cells: PHM analysis. A dose–response curve was generated by incubating the  iPAM cells with the indicated doses of Dox (micrograms per milliliter) for 48 h. NT and stably transfected pUHD−, pUHD+  (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines  were also analyzed. (A) PHM specific activity was measured in  cell extracts (mean ± SEM; n = 2–4). Western blots of 20 μg of  cell protein (B) and spent medium corresponding to 200 μg cell  protein (6 h basal collection, C) were visualized with antiserum to  PHM (Ab 1764). (D) Blots from cell extracts were digitized; expression in PAM-1 cells was set to 100%, and the relative contributions of intact 120-kD PAM-1 (open bars) and 46-kD PHM  (filled bars) are plotted as a stacked bar graph. (E) For each cell  line or dose of Dox-treated cells, the amount of 46-kD PHM in  cell extracts (B) or spent medium (C) (taking into account the 10-fold excess volume analyzed) was expressed as a percentage of  total PAM; total PAM = 120-kD PAM-1 (cells) + 46-kD PHM  (cells and media). Similar results were obtained in two additional  experiments.
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Figure 3: Induction of PAM protein in iPAM cells: PHM analysis. A dose–response curve was generated by incubating the iPAM cells with the indicated doses of Dox (micrograms per milliliter) for 48 h. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines were also analyzed. (A) PHM specific activity was measured in cell extracts (mean ± SEM; n = 2–4). Western blots of 20 μg of cell protein (B) and spent medium corresponding to 200 μg cell protein (6 h basal collection, C) were visualized with antiserum to PHM (Ab 1764). (D) Blots from cell extracts were digitized; expression in PAM-1 cells was set to 100%, and the relative contributions of intact 120-kD PAM-1 (open bars) and 46-kD PHM (filled bars) are plotted as a stacked bar graph. (E) For each cell line or dose of Dox-treated cells, the amount of 46-kD PHM in cell extracts (B) or spent medium (C) (taking into account the 10-fold excess volume analyzed) was expressed as a percentage of total PAM; total PAM = 120-kD PAM-1 (cells) + 46-kD PHM (cells and media). Similar results were obtained in two additional experiments.

Mentions: Since one of our goals was to examine cells expressing different levels of PAM-1, iPAM cells were treated for 48 h with increasing concentrations of Dox and compared with control cells using enzyme assays (Fig. 3 A) and Western blots (Fig. 3, B and C). Controls included nontransfected AtT-20 cells as well as AtT-20 cells expressing only the reverse tetracycline repressor (pUHD−, untreated; pUHD+, treated with 4 μg/ml Dox for 48 h) or expressing PAM-1 or PHM4. As with PAM mRNA (Fig. 2), Dox exerted a dose-dependent effect on PHM activity (Fig. 3 A). Peak levels of PHM specific activity were observed after treatment with 1–4 μg/ml of Dox. PHM specific activity in the noninduced iPAM cells was similar to nontransfected cells, untreated, and Dox-treated pUHD cells (Fig. 3 A). The PHM specific activity of maximally induced iPAM cells was 20–30-fold higher than noninduced iPAM cells and comparable to that of the stably transfected PAM-1 and PHM4 cells (Fig. 3 A). The sensitivity of iPAM cells to induction with low amounts of Dox can be seen by the two- to fivefold increase in PHM specific activity when noninduced cells are compared with cells treated with 0.0625 μg/ml 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)

Induction of PAM protein in iPAM cells: PHM analysis. A dose–response curve was generated by incubating the  iPAM cells with the indicated doses of Dox (micrograms per milliliter) for 48 h. NT and stably transfected pUHD−, pUHD+  (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines  were also analyzed. (A) PHM specific activity was measured in  cell extracts (mean ± SEM; n = 2–4). Western blots of 20 μg of  cell protein (B) and spent medium corresponding to 200 μg cell  protein (6 h basal collection, C) were visualized with antiserum to  PHM (Ab 1764). (D) Blots from cell extracts were digitized; expression in PAM-1 cells was set to 100%, and the relative contributions of intact 120-kD PAM-1 (open bars) and 46-kD PHM  (filled bars) are plotted as a stacked bar graph. (E) For each cell  line or dose of Dox-treated cells, the amount of 46-kD PHM in  cell extracts (B) or spent medium (C) (taking into account the 10-fold excess volume analyzed) was expressed as a percentage of  total PAM; total PAM = 120-kD PAM-1 (cells) + 46-kD PHM  (cells and media). Similar results were obtained in two additional  experiments.
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Figure 3: Induction of PAM protein in iPAM cells: PHM analysis. A dose–response curve was generated by incubating the iPAM cells with the indicated doses of Dox (micrograms per milliliter) for 48 h. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines were also analyzed. (A) PHM specific activity was measured in cell extracts (mean ± SEM; n = 2–4). Western blots of 20 μg of cell protein (B) and spent medium corresponding to 200 μg cell protein (6 h basal collection, C) were visualized with antiserum to PHM (Ab 1764). (D) Blots from cell extracts were digitized; expression in PAM-1 cells was set to 100%, and the relative contributions of intact 120-kD PAM-1 (open bars) and 46-kD PHM (filled bars) are plotted as a stacked bar graph. (E) For each cell line or dose of Dox-treated cells, the amount of 46-kD PHM in cell extracts (B) or spent medium (C) (taking into account the 10-fold excess volume analyzed) was expressed as a percentage of total PAM; total PAM = 120-kD PAM-1 (cells) + 46-kD PHM (cells and media). Similar results were obtained in two additional experiments.
Mentions: Since one of our goals was to examine cells expressing different levels of PAM-1, iPAM cells were treated for 48 h with increasing concentrations of Dox and compared with control cells using enzyme assays (Fig. 3 A) and Western blots (Fig. 3, B and C). Controls included nontransfected AtT-20 cells as well as AtT-20 cells expressing only the reverse tetracycline repressor (pUHD−, untreated; pUHD+, treated with 4 μg/ml Dox for 48 h) or expressing PAM-1 or PHM4. As with PAM mRNA (Fig. 2), Dox exerted a dose-dependent effect on PHM activity (Fig. 3 A). Peak levels of PHM specific activity were observed after treatment with 1–4 μg/ml of Dox. PHM specific activity in the noninduced iPAM cells was similar to nontransfected cells, untreated, and Dox-treated pUHD cells (Fig. 3 A). The PHM specific activity of maximally induced iPAM cells was 20–30-fold higher than noninduced iPAM cells and comparable to that of the stably transfected PAM-1 and PHM4 cells (Fig. 3 A). The sensitivity of iPAM cells to induction with low amounts of Dox can be seen by the two- to fivefold increase in PHM specific activity when noninduced cells are compared with cells treated with 0.0625 μg/ml 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.

Show MeSH
Related in: MedlinePlus