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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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Model for the effects of increased PAM expression in  AtT-20 cells. In noninduced iPAM cells (iPAM−) neuroendocrine-specific cleavages begin to occur in ISGs. Products are  stored in mature granules (MSGs). Addition of secretagogue  stimulates Ca2+ entry (mimicked here by addition of Ba2+) and  secretion from mature, but not from immature, secretory granules. Budding from ISGs allows nonaggregated content proteins  and membrane proteins to leave the ISG and undergo secretion  via constitutive-like vesicles (CLV) or return to the TGN. Filamentous actin is scattered throughout the cell. Upon expression  of PAM-1 (iPAM+ or PAM-1), the actin cytoskeleton is reorganized and concentrated more at the plasma membrane (PM).  Mature granules are no longer collected at the tips of cell processes, regulated exocytosis no longer occurs, and cleavage of  secretory granule content proteins is slowed. The interaction of  the cytosolic domain of PAM-1 with an endogenous Kalirin-like  protein and thus with Rac1 is postulated to cause the observed alterations in the actin cytoskeleton. Inhibition of regulated exocytosis and secretory granule maturation and localization may involve contributions from the lumenal domains of PAM as well as  the cytosolic domain.
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Figure 11: Model for the effects of increased PAM expression in AtT-20 cells. In noninduced iPAM cells (iPAM−) neuroendocrine-specific cleavages begin to occur in ISGs. Products are stored in mature granules (MSGs). Addition of secretagogue stimulates Ca2+ entry (mimicked here by addition of Ba2+) and secretion from mature, but not from immature, secretory granules. Budding from ISGs allows nonaggregated content proteins and membrane proteins to leave the ISG and undergo secretion via constitutive-like vesicles (CLV) or return to the TGN. Filamentous actin is scattered throughout the cell. Upon expression of PAM-1 (iPAM+ or PAM-1), the actin cytoskeleton is reorganized and concentrated more at the plasma membrane (PM). Mature granules are no longer collected at the tips of cell processes, regulated exocytosis no longer occurs, and cleavage of secretory granule content proteins is slowed. The interaction of the cytosolic domain of PAM-1 with an endogenous Kalirin-like protein and thus with Rac1 is postulated to cause the observed alterations in the actin cytoskeleton. Inhibition of regulated exocytosis and secretory granule maturation and localization may involve contributions from the lumenal domains of PAM as well as the cytosolic domain.

Mentions: The signals mediating the trafficking of soluble, lumenal proteins and membrane proteins from TGN into immature secretory granules (ISGs) and the maturation of secretory granules are not yet understood. Our observation that induction of PAM-1 expression causes dramatic changes in the actin cytoskeleton, eliminates regulated exocytosis, causes the relocalization of soluble lumenal proteins, and limits the cleavage of lumenal proteins, provides new insight into this process. PAM-1 has lumenal and cytosolic components, and both may contribute to the observed effects. We suggest two ways in which membrane PAM could affect the regulated secretory pathway: first, the COOH-terminal domain of PAM may interact with cytosolic factors involved in regulating microtubules and the actin cytoskeleton; second, the lumenal domains of PAM may aggregate with other lumenal proteins (Colomer et al., 1996) (Fig. 11).


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)

Model for the effects of increased PAM expression in  AtT-20 cells. In noninduced iPAM cells (iPAM−) neuroendocrine-specific cleavages begin to occur in ISGs. Products are  stored in mature granules (MSGs). Addition of secretagogue  stimulates Ca2+ entry (mimicked here by addition of Ba2+) and  secretion from mature, but not from immature, secretory granules. Budding from ISGs allows nonaggregated content proteins  and membrane proteins to leave the ISG and undergo secretion  via constitutive-like vesicles (CLV) or return to the TGN. Filamentous actin is scattered throughout the cell. Upon expression  of PAM-1 (iPAM+ or PAM-1), the actin cytoskeleton is reorganized and concentrated more at the plasma membrane (PM).  Mature granules are no longer collected at the tips of cell processes, regulated exocytosis no longer occurs, and cleavage of  secretory granule content proteins is slowed. The interaction of  the cytosolic domain of PAM-1 with an endogenous Kalirin-like  protein and thus with Rac1 is postulated to cause the observed alterations in the actin cytoskeleton. Inhibition of regulated exocytosis and secretory granule maturation and localization may involve contributions from the lumenal domains of PAM as well as  the cytosolic domain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132922&req=5

Figure 11: Model for the effects of increased PAM expression in AtT-20 cells. In noninduced iPAM cells (iPAM−) neuroendocrine-specific cleavages begin to occur in ISGs. Products are stored in mature granules (MSGs). Addition of secretagogue stimulates Ca2+ entry (mimicked here by addition of Ba2+) and secretion from mature, but not from immature, secretory granules. Budding from ISGs allows nonaggregated content proteins and membrane proteins to leave the ISG and undergo secretion via constitutive-like vesicles (CLV) or return to the TGN. Filamentous actin is scattered throughout the cell. Upon expression of PAM-1 (iPAM+ or PAM-1), the actin cytoskeleton is reorganized and concentrated more at the plasma membrane (PM). Mature granules are no longer collected at the tips of cell processes, regulated exocytosis no longer occurs, and cleavage of secretory granule content proteins is slowed. The interaction of the cytosolic domain of PAM-1 with an endogenous Kalirin-like protein and thus with Rac1 is postulated to cause the observed alterations in the actin cytoskeleton. Inhibition of regulated exocytosis and secretory granule maturation and localization may involve contributions from the lumenal domains of PAM as well as the cytosolic domain.
Mentions: The signals mediating the trafficking of soluble, lumenal proteins and membrane proteins from TGN into immature secretory granules (ISGs) and the maturation of secretory granules are not yet understood. Our observation that induction of PAM-1 expression causes dramatic changes in the actin cytoskeleton, eliminates regulated exocytosis, causes the relocalization of soluble lumenal proteins, and limits the cleavage of lumenal proteins, provides new insight into this process. PAM-1 has lumenal and cytosolic components, and both may contribute to the observed effects. We suggest two ways in which membrane PAM could affect the regulated secretory pathway: first, the COOH-terminal domain of PAM may interact with cytosolic factors involved in regulating microtubules and the actin cytoskeleton; second, the lumenal domains of PAM may aggregate with other lumenal proteins (Colomer et al., 1996) (Fig. 11).

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