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Originally published In Press as doi:10.1074/jbc.M212128200 on February 12, 2003

J. Biol. Chem., Vol. 278, Issue 17, 15007-15014, April 25, 2003
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Biological Processing of the Cocaine and Amphetamine-regulated Transcript Precursors by Prohormone Convertases, PC2 and PC1/3*

Arunangsu DeyDagger , Xiaorong XhuDagger , Raymond Carroll§, Christopher W. Turck, Jeffrey SteinDagger , and Donald F. SteinerDagger §||

From the Dagger  Department of Biochemistry and Molecular Biology, the § Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637 and the  Howard Hughes Medical Institute, University of California, San Francisco, California 94143

Cocaine and amphetamine-regulated transcript (CART), a neuroendocrine peptide influencing reward, feeding/appetite, and stress responses is derived from two peptide precursors of 129 and 116 amino acid (aa) residues that arise via alternative splicing from a single Cart gene in rats and mice. The signal peptide constitutes the first 27 aa resulting in pro-CART molecules of either 102 or 89 aa. In the present study, we have shown that pro-CART is a substrate for the neuroendocrine subtilisin/kexin-like prohormone convertases, PC2 (SPC2) and PC1/3 (SPC3). By using different neuroendocrine cell lines, with or without endogenous expression of either PC2 or PC1/3 or both enzymes, we have demonstrated through transient transfection studies that long pro-CART gives rise to an intermediate peptide, residues 33-102, and the two major bioactive CART forms, residues 55-102 (I) and 62-102 (II), respectively. Likewise, short pro-CART also generates three peptides, an intermediate, residues 10-89, and the two identical bioactive CART forms. We have confirmed the identities of the bioactive and intermediate CART molecules by microsequencing and/or high performance liquid chromatography and mass spectrometry. We have shown that PC2 is more efficient in generating bioactive CART I compared with PC1/3, whereas the production of the smaller bioactive CART II is exclusively carried out by PC2. PC1/3 is predominantly responsible for generating the intermediate CART fragments, 33-102 and 10-89, from long and short pro-CART, respectively. To compare in vitro and in vivo processing of pro-CART, we have examined its processing in PC2, 7B2, and PC1/3 knock-out mouse hypothalamic extracts and demonstrated that, as in vitro, PC2 is more potent than PC1/3 in generating bioactive CART I whereas bioactive CART II is solely generated by PC2. Also, in vivo, we have shown that PC1/3 is predominantly active in liberating the two intermediate CART fragments, 33-102 and 10-89. These findings confirm the key roles of PC2 and PC1/3 acting together or separately to carry out CART processing in selected sites in vivo.

* This work was supported by National Institutes of Health Grants DK13914 and DK20595 and the Howard Hughes Medical Institute.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.

|| To whom correspondence should be addressed. Tel.: 773-702-1334; Fax: 773-702-4292; E-mail: dfsteine@midway.uchicago.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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