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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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Effect of PAM expression on PC1. (A) Structure of  preproPC1; preproPC1 has a signal peptide (SP), proregion  (Pro), subtilisin-like catalytic domain (Subtilisin), P-domain (P),  and COOH-terminal domain (COOH). ProPC1 (87 kD) is  cleaved to yield PC1 (82 kD) and PC1ΔC (66 kD). iPAM cells  were treated with the indicated doses of Dox for 48 h. Western  blots of 20 μg of cell extracts (B) and spent medium (not shown)  were visualized with antiserum to PC1 (Ab JH888). (C) Blots  were digitized. (D) Immunofluorescent staining of NT, iPAM−,  iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4  cells was viewed using phase optics and stained with an antibody  for PC1 (Ab JH888). Bar, 10 μm.
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Figure 9: Effect of PAM expression on PC1. (A) Structure of preproPC1; preproPC1 has a signal peptide (SP), proregion (Pro), subtilisin-like catalytic domain (Subtilisin), P-domain (P), and COOH-terminal domain (COOH). ProPC1 (87 kD) is cleaved to yield PC1 (82 kD) and PC1ΔC (66 kD). iPAM cells were treated with the indicated doses of Dox for 48 h. Western blots of 20 μg of cell extracts (B) and spent medium (not shown) were visualized with antiserum to PC1 (Ab JH888). (C) Blots were digitized. (D) Immunofluorescent staining of NT, iPAM−, iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cells was viewed using phase optics and stained with an antibody for PC1 (Ab JH888). Bar, 10 μm.

Mentions: To ask whether expression of membrane PAM might affect other LDCV proteins in addition to ACTH, PC1 was investigated. The major products of proPC1 cleavage are mature 82-kD PC1, produced rapidly in the endoplasmic reticulum, and 66-kD PC1ΔC, produced only in LDCVs (Fig. 9 A; Zhou and Mains, 1994). A dose–response curve for Dox induction of PAM-1 expression was generated as described in Figs. 2–4 and cell extracts were subjected to Western blot analysis with antibodies to PC1 (Fig. 9 B). In nontransfected, PHM4, and pUHD− or pUHD+ cells, PC1ΔC was the major form of PC1 in cell extracts. In contrast, in PAM-1 cells, mature 82-kD PC1 predominated in cell extracts. Increasing PAM-1 expression in iPAM cells caused a dose-dependent decrease in the production of PC1ΔC from mature PC1; whereas 65% of the total PC1 was PC1ΔC in noninduced cells, only 30% was PC1ΔC in the maximally induced iPAM cells (Fig. 9 C). PC1ΔC accounted for even less of the total PC1 in stably transfected PAM-1 cells.


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)

Effect of PAM expression on PC1. (A) Structure of  preproPC1; preproPC1 has a signal peptide (SP), proregion  (Pro), subtilisin-like catalytic domain (Subtilisin), P-domain (P),  and COOH-terminal domain (COOH). ProPC1 (87 kD) is  cleaved to yield PC1 (82 kD) and PC1ΔC (66 kD). iPAM cells  were treated with the indicated doses of Dox for 48 h. Western  blots of 20 μg of cell extracts (B) and spent medium (not shown)  were visualized with antiserum to PC1 (Ab JH888). (C) Blots  were digitized. (D) Immunofluorescent staining of NT, iPAM−,  iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4  cells was viewed using phase optics and stained with an antibody  for PC1 (Ab JH888). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 9: Effect of PAM expression on PC1. (A) Structure of preproPC1; preproPC1 has a signal peptide (SP), proregion (Pro), subtilisin-like catalytic domain (Subtilisin), P-domain (P), and COOH-terminal domain (COOH). ProPC1 (87 kD) is cleaved to yield PC1 (82 kD) and PC1ΔC (66 kD). iPAM cells were treated with the indicated doses of Dox for 48 h. Western blots of 20 μg of cell extracts (B) and spent medium (not shown) were visualized with antiserum to PC1 (Ab JH888). (C) Blots were digitized. (D) Immunofluorescent staining of NT, iPAM−, iPAM+ (treated with 4 μg/ml Dox for 48 h), PAM-1, and PHM4 cells was viewed using phase optics and stained with an antibody for PC1 (Ab JH888). Bar, 10 μm.
Mentions: To ask whether expression of membrane PAM might affect other LDCV proteins in addition to ACTH, PC1 was investigated. The major products of proPC1 cleavage are mature 82-kD PC1, produced rapidly in the endoplasmic reticulum, and 66-kD PC1ΔC, produced only in LDCVs (Fig. 9 A; Zhou and Mains, 1994). A dose–response curve for Dox induction of PAM-1 expression was generated as described in Figs. 2–4 and cell extracts were subjected to Western blot analysis with antibodies to PC1 (Fig. 9 B). In nontransfected, PHM4, and pUHD− or pUHD+ cells, PC1ΔC was the major form of PC1 in cell extracts. In contrast, in PAM-1 cells, mature 82-kD PC1 predominated in cell extracts. Increasing PAM-1 expression in iPAM cells caused a dose-dependent decrease in the production of PC1ΔC from mature PC1; whereas 65% of the total PC1 was PC1ΔC in noninduced cells, only 30% was PC1ΔC in the maximally induced iPAM cells (Fig. 9 C). PC1ΔC accounted for even less of the total PC1 in stably transfected PAM-1 cells.

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