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Papers In Press, published online ahead of print February 12, 2003
Dept. of Biochem. & Molec. Biology, H.H.M.I., Univ. of Chicago, Chicago, IL 60637
Corresponding Author: dfsteine{at}midway.uchicago.edu
Cocaine and amphetamine regulated transcript (CART), a neuroendocrine peptide inhibiting feeding/appetite, encodes two peptides of 129 and 116 amino acid (aa) residues in rats and mice. The signal peptide constitutes the first 27 aa resulting in proCART molecules of either 102 or 89 aa. In the present study, we have shown that proCART is a substrate for the subtilisin/kexin-like prohormone convertases, PC2 (SPC2) and PC3/PC1 (SPC3). By using different neuroendocrine cell lines, with or without endogenous expression of either PC2 or PC3 or both enzymes, we have demonstrated through transient transfection studies and microsequencing analysis that two major peptides (residues 33-102 and 55-102) are processed from long proCART, whereas short proCART generates a single major peptide (residues 42-89, equivalent to residues 55-102 of the long form). We have shown that PC2 is more efficient in generating the bioactive form of CART, aa 55-102 / 42-89, compared to PC3. Employing HPLC and mass spectrometry, we have confirmed the identity of this bioactive CART fragment. We have also demonstrated the presence of the shorter 49-89 active fragment by generating tagged proCART (short) with Flag epitope at its C-terminus. Pulse-chase studies have shown the efficient secretion of all the processed peptides along with the proCART molecules. In addition we have shown that proCART is posttranslationally modified through sulfation. To compare in vitro and in vivo processing of proCART, we have analyzed the defects in proCART processing in PC2, PC3/PC1 and 7B2 knock-out mouse hypothalamic extracts and demonstrated that PC2 is more potent than PC3 in generating bioactive CART while PC3 is more efficient in generating an intermediate form from long proCART.
J. Biol. Chem, 10.1074/jbc.M212128200
Submitted on November 27, 2002
Revised on February 11, 2003
Accepted on February 12, 2003
Biological processing of the CART precursor by prohormone convertases PC2 and PC1/PC3
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