Biological processing of the cocaine and amphetamine-regulated transcript precursors by prohormone convertases, PC2 and PC1/3.
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.
Dey
Arunangsu
A
Department of Biochemistry and Molecular Biology, the Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA.
Xhu
Xiaorong
X
Carroll
Raymond
R
Turck
Christopher W
CW
Stein
Jeffrey
J
Steiner
Donald F
DF
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007031:Hypothalamus / Q000737:chemistry / Q000166:cytology; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; C473668:Pcsk2 protein, mouse; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases