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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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Structures of PAM proteins studied. Intact PAM-1 is  an integral membrane protein of 120 kD that consists of two catalytic domains (PHM and PAL) which are separated by the noncatalytic exon A region (hatched box) (Eipper et al., 1993). A  proteolytic cleavage site is present within exon A. The PHM and  PAL catalytic domains lie within the lumenal compartment. The  major protein products generated from PAM-1 cleavage in neuroendocrine cells are 46-kD PHM (soluble) and 70-kD PAL  (membrane bound, PALm); PALm includes a transmembrane  domain (TMD) and a cytoplasmic domain (CD). PHM4 is a soluble, naturally occurring, alternatively spliced variant of PAM  (previously called PAM-4); PHM4 contains the PHM and exon A  domains followed by a unique COOH terminus. AtT-20 cells stably transfected with vectors encoding PAM-1 and PHM4 were  examined. The specificities of the PAM antisera used in this  study are shown. mAb, mouse monoclonal antibody; Ab, rabbit  polyclonal antibody.
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Figure 1: Structures of PAM proteins studied. Intact PAM-1 is an integral membrane protein of 120 kD that consists of two catalytic domains (PHM and PAL) which are separated by the noncatalytic exon A region (hatched box) (Eipper et al., 1993). A proteolytic cleavage site is present within exon A. The PHM and PAL catalytic domains lie within the lumenal compartment. The major protein products generated from PAM-1 cleavage in neuroendocrine cells are 46-kD PHM (soluble) and 70-kD PAL (membrane bound, PALm); PALm includes a transmembrane domain (TMD) and a cytoplasmic domain (CD). PHM4 is a soluble, naturally occurring, alternatively spliced variant of PAM (previously called PAM-4); PHM4 contains the PHM and exon A domains followed by a unique COOH terminus. AtT-20 cells stably transfected with vectors encoding PAM-1 and PHM4 were examined. The specificities of the PAM antisera used in this study are shown. mAb, mouse monoclonal antibody; Ab, rabbit polyclonal antibody.

Mentions: Peptidylglycine α-amidating monooxygenase (PAM)1 is a bifunctional enzyme found in nearly all large dense core vesicles (LDCVs; Eipper et al., 1993). The major forms of PAM are type I integral membrane proteins that catalyze the COOH-terminal amidation of glycine-extended peptides in a two-step process (Eipper et al., 1993; Kolhekar et al., 1997). Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction, whereas peptidyl-α-hydroxyglycine α-amidating lyase (PAL) catalyzes the second step. The precursor PAM protein, PAM-1 (Fig. 1), is composed of an initial signal and propeptide sequence followed by the PHM catalytic domain, a noncatalytic domain referred to as exon A, the PAL catalytic domain, a transmembrane domain, and a COOH-terminal domain (Yun et al., 1993). The two catalytic domains of PAM can be expressed independently as soluble PHM and PAL, and both domains have been shown to be targeted efficiently to LDCVs (Milgram et al., 1992). In contrast, when integral membrane forms of PAM were expressed in an AtT-20 mouse corticotrope cell line, they were predominantly localized to the trans-Golgi network (TGN) (Milgram et al., 1993, 1994, 1997). The COOH-terminal domain of PAM interacts with proteins that could affect the cytoskeletal organization via their effects on actin and tubulin (Alam et al., 1997).


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)

Structures of PAM proteins studied. Intact PAM-1 is  an integral membrane protein of 120 kD that consists of two catalytic domains (PHM and PAL) which are separated by the noncatalytic exon A region (hatched box) (Eipper et al., 1993). A  proteolytic cleavage site is present within exon A. The PHM and  PAL catalytic domains lie within the lumenal compartment. The  major protein products generated from PAM-1 cleavage in neuroendocrine cells are 46-kD PHM (soluble) and 70-kD PAL  (membrane bound, PALm); PALm includes a transmembrane  domain (TMD) and a cytoplasmic domain (CD). PHM4 is a soluble, naturally occurring, alternatively spliced variant of PAM  (previously called PAM-4); PHM4 contains the PHM and exon A  domains followed by a unique COOH terminus. AtT-20 cells stably transfected with vectors encoding PAM-1 and PHM4 were  examined. The specificities of the PAM antisera used in this  study are shown. mAb, mouse monoclonal antibody; Ab, rabbit  polyclonal antibody.
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Related In: Results  -  Collection

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Figure 1: Structures of PAM proteins studied. Intact PAM-1 is an integral membrane protein of 120 kD that consists of two catalytic domains (PHM and PAL) which are separated by the noncatalytic exon A region (hatched box) (Eipper et al., 1993). A proteolytic cleavage site is present within exon A. The PHM and PAL catalytic domains lie within the lumenal compartment. The major protein products generated from PAM-1 cleavage in neuroendocrine cells are 46-kD PHM (soluble) and 70-kD PAL (membrane bound, PALm); PALm includes a transmembrane domain (TMD) and a cytoplasmic domain (CD). PHM4 is a soluble, naturally occurring, alternatively spliced variant of PAM (previously called PAM-4); PHM4 contains the PHM and exon A domains followed by a unique COOH terminus. AtT-20 cells stably transfected with vectors encoding PAM-1 and PHM4 were examined. The specificities of the PAM antisera used in this study are shown. mAb, mouse monoclonal antibody; Ab, rabbit polyclonal antibody.
Mentions: Peptidylglycine α-amidating monooxygenase (PAM)1 is a bifunctional enzyme found in nearly all large dense core vesicles (LDCVs; Eipper et al., 1993). The major forms of PAM are type I integral membrane proteins that catalyze the COOH-terminal amidation of glycine-extended peptides in a two-step process (Eipper et al., 1993; Kolhekar et al., 1997). Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction, whereas peptidyl-α-hydroxyglycine α-amidating lyase (PAL) catalyzes the second step. The precursor PAM protein, PAM-1 (Fig. 1), is composed of an initial signal and propeptide sequence followed by the PHM catalytic domain, a noncatalytic domain referred to as exon A, the PAL catalytic domain, a transmembrane domain, and a COOH-terminal domain (Yun et al., 1993). The two catalytic domains of PAM can be expressed independently as soluble PHM and PAL, and both domains have been shown to be targeted efficiently to LDCVs (Milgram et al., 1992). In contrast, when integral membrane forms of PAM were expressed in an AtT-20 mouse corticotrope cell line, they were predominantly localized to the trans-Golgi network (TGN) (Milgram et al., 1993, 1994, 1997). The COOH-terminal domain of PAM interacts with proteins that could affect the cytoskeletal organization via their effects on actin and tubulin (Alam et al., 1997).

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