title: An amphipathic alpha-helix in the prodomain of cocaine and amphetamine regulated transcript peptide precursor serves as its sorting signal to the regulated secretory pathway.
abstract: Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1-41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24-37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.
authors.affiliation: Millennium Science Nucleus in Stress and Addiction, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile. eblanco@uc.cl
authors.forename: Elías H
authors.initials: EH
authors.lastname: Blanco
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Lagos
authors.forename: María Estela
authors.initials: ME
authors.lastname: Andrés
authors.forename: Katia
authors.initials: K
authors.lastname: Gysling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D016716:PC12 Cells; D011498:Protein Precursors / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D014162:Transfection; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
chemical_list: D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins