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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: PAL analysis.  (A) PAL specific activity was measured in cell extracts prepared  as described in Fig. 3 (mean ± SEM; n = 2–4). (B) Western blots  were carried out on cell extracts using antisera to PAL. (C) Blots  were digitized; expression in PAM-1 cells was set to 100% and  the relative amounts of PAM-1 (open bars) and 70-kD PALm  (filled bars) are plotted as a stacked bar graph. (D) For each cell  line or dose of Dox-treated cells, the amount of 70-kD PAL in  the cell extract is expressed as a percentage of the total PAM protein in the extract; total PAM = 120-kD PAM-1 (cells) + 70-kD  PAL (cells). Similar results were obtained in two additional experiments.
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Figure 4: Induction of PAM protein in iPAM cells: PAL analysis. (A) PAL specific activity was measured in cell extracts prepared as described in Fig. 3 (mean ± SEM; n = 2–4). (B) Western blots were carried out on cell extracts using antisera to PAL. (C) Blots were digitized; expression in PAM-1 cells was set to 100% and the relative amounts of PAM-1 (open bars) and 70-kD PALm (filled bars) are plotted as a stacked bar graph. (D) For each cell line or dose of Dox-treated cells, the amount of 70-kD PAL in the cell extract is expressed as a percentage of the total PAM protein in the extract; total PAM = 120-kD PAM-1 (cells) + 70-kD PAL (cells). Similar results were obtained in two additional experiments.

Mentions: As with the PAM mRNA, PHM protein expression, and PHM enzyme activity, a dose-dependent effect of Dox on PAL enzyme activity was observed (Fig. 4 A). Increased expression of PAL activity was apparent with the lowest doses of Dox tested. Peak levels of PAL specific activity were observed after treatment with 2–4 μg/ml Dox; treatment with 8 μg/ml Dox resulted in decreased PAL activity. PAL specific activity in the noninduced iPAM cells was similar to nontransfected, untreated, and Dox-treated pUHD cells (Fig. 4 A). The PAL specific activity of maximally induced iPAM cells was comparable to that of the stably transfected PAM-1 cells and two- to threefold higher than PHM specific activity, reflecting the higher enzymatic turnover rate of PAL (Eipper et al., 1993).


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: PAL analysis.  (A) PAL specific activity was measured in cell extracts prepared  as described in Fig. 3 (mean ± SEM; n = 2–4). (B) Western blots  were carried out on cell extracts using antisera to PAL. (C) Blots  were digitized; expression in PAM-1 cells was set to 100% and  the relative amounts of PAM-1 (open bars) and 70-kD PALm  (filled bars) are plotted as a stacked bar graph. (D) For each cell  line or dose of Dox-treated cells, the amount of 70-kD PAL in  the cell extract is expressed as a percentage of the total PAM protein in the extract; total PAM = 120-kD PAM-1 (cells) + 70-kD  PAL (cells). Similar results were obtained in two additional experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132922&req=5

Figure 4: Induction of PAM protein in iPAM cells: PAL analysis. (A) PAL specific activity was measured in cell extracts prepared as described in Fig. 3 (mean ± SEM; n = 2–4). (B) Western blots were carried out on cell extracts using antisera to PAL. (C) Blots were digitized; expression in PAM-1 cells was set to 100% and the relative amounts of PAM-1 (open bars) and 70-kD PALm (filled bars) are plotted as a stacked bar graph. (D) For each cell line or dose of Dox-treated cells, the amount of 70-kD PAL in the cell extract is expressed as a percentage of the total PAM protein in the extract; total PAM = 120-kD PAM-1 (cells) + 70-kD PAL (cells). Similar results were obtained in two additional experiments.
Mentions: As with the PAM mRNA, PHM protein expression, and PHM enzyme activity, a dose-dependent effect of Dox on PAL enzyme activity was observed (Fig. 4 A). Increased expression of PAL activity was apparent with the lowest doses of Dox tested. Peak levels of PAL specific activity were observed after treatment with 2–4 μg/ml Dox; treatment with 8 μg/ml Dox resulted in decreased PAL activity. PAL specific activity in the noninduced iPAM cells was similar to nontransfected, untreated, and Dox-treated pUHD cells (Fig. 4 A). The PAL specific activity of maximally induced iPAM cells was comparable to that of the stably transfected PAM-1 cells and two- to threefold higher than PHM specific activity, reflecting the higher enzymatic turnover rate of PAL (Eipper et al., 1993).

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