Localization of Metallocarboxypeptidase D in AtT-20 Cells
POTENTIAL ROLE IN PROHORMONE PROCESSING*
- From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Abstract
Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in thetrans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.
Footnotes
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↵* This work was supported by National Institutes of Health Grant R01 DK-51271 and by Research Scientist Development K02 Award DA-00194 (to L. D. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-430-8954; E-mail:fricker{at}aecom.yu.edu.
- Abbreviations:
- PC
-
prohormone convertase
- CPE
-
carboxypeptidase E
- CPD
-
carboxypeptidase D
- PAM
-
peptidylglycine-α-amidating monooxygenase
- TGN
-
trans-Golgi network
- ACTH
-
adrenocorticotrophic hormone
- POMC
-
proopiomelanocortin
- PBS
-
phosphate-buffered saline
- ER
-
endoplasmic reticulum
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- Received October 19, 1998.
- Revision received February 10, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
