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

Elías H Blanco,Carlos F Lagos,María Estela Andrés,Katia Gysling,Anna Tramontano

doi:10.1371/journal.pone.0059695

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.

Highlights

Cocaine and Amphetamine Regulated Transcript (CART) was identified as an mRNA that increases its level in rat striatum after binge acute doses of cocaine or amphetamine [1]
In order to identify the proCART sorting domain and the effect of the L34F mutation in proCART sorting, we studied the subcellular localization of the full-length or truncated forms of PLOS ONE | www.plosone.org proCART Sorting Domain human proCART fused to monomeric EGFP (EGFPm) expressed in PC12 cells
This alternative splicing described yields an isoform of proCART with an additional 13 amino acids inserted between positions 26–27

Summary

Introduction

Cocaine and Amphetamine Regulated Transcript (CART) was identified as an mRNA that increases its level in rat striatum after binge acute doses of cocaine or amphetamine [1]. Central injection of CART42–89 neuropeptide in the rodent brain increases anxiogenic behavior [2] and suppresses appetite [3,4]. Central injection of CART42–89 neuropeptide inhibits the increase in appetite triggered by neuropeptide Y (NPY), a potent orexigenic neuropeptide [3,5]. The CART neuropeptide precursor (proCART) of 89 residues is sorted and stored in secretory granules, a hallmark of the regulated secretory pathway [7]. PC1/3 and PC2 enter the regulated secretory pathway and are stored in secretory granules [11]. The processing of proCART yields two bioactive CART neuropeptides, CART42–89 and CART49–89 [12,13]. The sorting of proCART into secretory granules is essential for its bioactivation. The mechanism by which proCART is sorted toward the secretory granules remains unknown

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Conclusion