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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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Biosynthesis of PAM after Dox induction. Identical  wells of iPAM cells were treated with 0.25 or 2.0 μg/ml Dox for  48 h and then incubated with [35S]Met for 30 min and either harvested immediately (pulse, P) or chased for 0.5, 2, or 4 h (chase);  for chase samples, cell extracts (C) and media (M) were analyzed. PAM proteins were immunoprecipitated using antisera to  PHM or PAL (not shown); only data for PHM immunoprecipitates from the pulse and 4-h chase samples are shown. (Inset)  Longer exposures of films of chase samples are shown.
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Figure 5: Biosynthesis of PAM after Dox induction. Identical wells of iPAM cells were treated with 0.25 or 2.0 μg/ml Dox for 48 h and then incubated with [35S]Met for 30 min and either harvested immediately (pulse, P) or chased for 0.5, 2, or 4 h (chase); for chase samples, cell extracts (C) and media (M) were analyzed. PAM proteins were immunoprecipitated using antisera to PHM or PAL (not shown); only data for PHM immunoprecipitates from the pulse and 4-h chase samples are shown. (Inset) Longer exposures of films of chase samples are shown.

Mentions: The alterations in steady-state levels of PHM and PAL with increasing levels of PAM expression could reflect differences in the rate of biosynthesis of PAM-1, cleavage of PAM-1, degradation, and/or secretion of PHM and PAL. To distinguish these possibilities, identical wells of iPAM cells treated with a low dose of Dox (0.25 μg/ml) or a high dose of Dox (2.0 μg/ml) were incubated in medium containing [35S]Met for 30 min and either harvested immediately (pulse) or chased in medium containing unlabeled Met (chase). PAM proteins immunoprecipitated from cell extracts and media were fractionated by SDS-PAGE (Fig. 5). Consistent with the steady-state levels of PHM and PAL protein and enzyme activity (Figs. 3 and 4), the rate of PAM-1 biosynthesis was 2.8 ± 0.4-fold higher (P < 0.005; n = 4) in cells induced with the high dose of Dox than in cells induced with the low dose of 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)

Biosynthesis of PAM after Dox induction. Identical  wells of iPAM cells were treated with 0.25 or 2.0 μg/ml Dox for  48 h and then incubated with [35S]Met for 30 min and either harvested immediately (pulse, P) or chased for 0.5, 2, or 4 h (chase);  for chase samples, cell extracts (C) and media (M) were analyzed. PAM proteins were immunoprecipitated using antisera to  PHM or PAL (not shown); only data for PHM immunoprecipitates from the pulse and 4-h chase samples are shown. (Inset)  Longer exposures of films of chase samples are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132922&req=5

Figure 5: Biosynthesis of PAM after Dox induction. Identical wells of iPAM cells were treated with 0.25 or 2.0 μg/ml Dox for 48 h and then incubated with [35S]Met for 30 min and either harvested immediately (pulse, P) or chased for 0.5, 2, or 4 h (chase); for chase samples, cell extracts (C) and media (M) were analyzed. PAM proteins were immunoprecipitated using antisera to PHM or PAL (not shown); only data for PHM immunoprecipitates from the pulse and 4-h chase samples are shown. (Inset) Longer exposures of films of chase samples are shown.
Mentions: The alterations in steady-state levels of PHM and PAL with increasing levels of PAM expression could reflect differences in the rate of biosynthesis of PAM-1, cleavage of PAM-1, degradation, and/or secretion of PHM and PAL. To distinguish these possibilities, identical wells of iPAM cells treated with a low dose of Dox (0.25 μg/ml) or a high dose of Dox (2.0 μg/ml) were incubated in medium containing [35S]Met for 30 min and either harvested immediately (pulse) or chased in medium containing unlabeled Met (chase). PAM proteins immunoprecipitated from cell extracts and media were fractionated by SDS-PAGE (Fig. 5). Consistent with the steady-state levels of PHM and PAL protein and enzyme activity (Figs. 3 and 4), the rate of PAM-1 biosynthesis was 2.8 ± 0.4-fold higher (P < 0.005; n = 4) in cells induced with the high dose of Dox than in cells induced with the low dose of 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