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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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Expression of PAM-1 alters secretion  of ACTH in AtT-20 cells. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml  Dox for 48 h), iPAM−, iPAM+ (treated with 4  μg/ml Dox for 48 h), PAM-1, and PHM4 cell  lines were analyzed as described in Materials and  Methods. (A) Medium was collected after two  30-min periods of basal secretion followed by a  30-min period of stimulated secretion (1 mM  BaCl2) and the levels of ACTH were determined. (B) Cells were extracted for measurement of total protein and immunoreactive  ACTH. Levels of ACTH from duplicate cultures  were measured in triplicate (A and B). (C) Medium was collected as in A and assayed for PHM  activity. Bars are mean ± SEM.
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Figure 8: Expression of PAM-1 alters secretion of ACTH in AtT-20 cells. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml Dox for 48 h), iPAM−, iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines were analyzed as described in Materials and Methods. (A) Medium was collected after two 30-min periods of basal secretion followed by a 30-min period of stimulated secretion (1 mM BaCl2) and the levels of ACTH were determined. (B) Cells were extracted for measurement of total protein and immunoreactive ACTH. Levels of ACTH from duplicate cultures were measured in triplicate (A and B). (C) Medium was collected as in A and assayed for PHM activity. Bars are mean ± SEM.

Mentions: Observing that the ACTH immunostaining patterns were markedly different in nontransfected cells and cells expressing PAM-1 (Figs. 6 C and 7), their basal and stimulated secretions of ACTH were examined. Secretion of ACTH was quantified using an immunoassay specific for the COOH terminus of ACTH(1-39); secretion was examined during two sequential basal collections and cells were then stimulated with 1 mM BaCl2 (Fig. 8 A). BaCl2 mimics Ca2+ and several secretagogues and supports a steady high rate of secretion (Mains and Eipper, 1984). As expected, in nontransfected AtT-20 cells, the addition of secretagogue to the medium stimulated ACTH secretion three- to fourfold above basal levels. A similar fold stimulation of ACTH secretion was observed in control pUHD cells whether or not they were treated with Dox (pUHD−, pUHD+) and in noninduced iPAM cells; none of these cells express exogenous PAM-1. In contrast, in iPAM cells treated with Dox to induce PAM-1 expression, addition of secretagogue to the medium did not stimulate ACTH secretion above basal levels (Fig. 8 A). The effects seen with the iPAM cells cannot be attributable to the Dox treatment alone since the pUHD+ cells responded to secretagogue as well as nontransfected AtT-20 cells or untreated pUHD− cells. As for Dox-treated iPAM cells, secretion of ACTH from stably transfected PAM-1 cells was not stimulated by secretagogue (Fig. 8 A). In contrast, AtT-20 cells expressing a soluble PAM protein (PHM4), exhibited a threefold increase in ACTH secretion in response to secretagogue. Treatment of the iPAM cells with a low, but effective dose of Dox (0.0625 μg/ml) resulted in a partial inhibition of the ability of secretagogue to stimulate ACTH secretion (data not shown). Thus, overexpression of PAM-1 is causally related to the inability of Dox-treated iPAM cells and PAM-1 cells to respond to secretagogue treatment.


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)

Expression of PAM-1 alters secretion  of ACTH in AtT-20 cells. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml  Dox for 48 h), iPAM−, iPAM+ (treated with 4  μg/ml Dox for 48 h), PAM-1, and PHM4 cell  lines were analyzed as described in Materials and  Methods. (A) Medium was collected after two  30-min periods of basal secretion followed by a  30-min period of stimulated secretion (1 mM  BaCl2) and the levels of ACTH were determined. (B) Cells were extracted for measurement of total protein and immunoreactive  ACTH. Levels of ACTH from duplicate cultures  were measured in triplicate (A and B). (C) Medium was collected as in A and assayed for PHM  activity. Bars are mean ± SEM.
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Related In: Results  -  Collection

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Figure 8: Expression of PAM-1 alters secretion of ACTH in AtT-20 cells. NT and stably transfected pUHD−, pUHD+ (treated with 4 μg/ml Dox for 48 h), iPAM−, iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cell lines were analyzed as described in Materials and Methods. (A) Medium was collected after two 30-min periods of basal secretion followed by a 30-min period of stimulated secretion (1 mM BaCl2) and the levels of ACTH were determined. (B) Cells were extracted for measurement of total protein and immunoreactive ACTH. Levels of ACTH from duplicate cultures were measured in triplicate (A and B). (C) Medium was collected as in A and assayed for PHM activity. Bars are mean ± SEM.
Mentions: Observing that the ACTH immunostaining patterns were markedly different in nontransfected cells and cells expressing PAM-1 (Figs. 6 C and 7), their basal and stimulated secretions of ACTH were examined. Secretion of ACTH was quantified using an immunoassay specific for the COOH terminus of ACTH(1-39); secretion was examined during two sequential basal collections and cells were then stimulated with 1 mM BaCl2 (Fig. 8 A). BaCl2 mimics Ca2+ and several secretagogues and supports a steady high rate of secretion (Mains and Eipper, 1984). As expected, in nontransfected AtT-20 cells, the addition of secretagogue to the medium stimulated ACTH secretion three- to fourfold above basal levels. A similar fold stimulation of ACTH secretion was observed in control pUHD cells whether or not they were treated with Dox (pUHD−, pUHD+) and in noninduced iPAM cells; none of these cells express exogenous PAM-1. In contrast, in iPAM cells treated with Dox to induce PAM-1 expression, addition of secretagogue to the medium did not stimulate ACTH secretion above basal levels (Fig. 8 A). The effects seen with the iPAM cells cannot be attributable to the Dox treatment alone since the pUHD+ cells responded to secretagogue as well as nontransfected AtT-20 cells or untreated pUHD− cells. As for Dox-treated iPAM cells, secretion of ACTH from stably transfected PAM-1 cells was not stimulated by secretagogue (Fig. 8 A). In contrast, AtT-20 cells expressing a soluble PAM protein (PHM4), exhibited a threefold increase in ACTH secretion in response to secretagogue. Treatment of the iPAM cells with a low, but effective dose of Dox (0.0625 μg/ml) resulted in a partial inhibition of the ability of secretagogue to stimulate ACTH secretion (data not shown). Thus, overexpression of PAM-1 is causally related to the inability of Dox-treated iPAM cells and PAM-1 cells to respond to secretagogue treatment.

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