Unacylated ghrelin binds heparan-sulfate proteoglycans which modulate its function

in Journal of Molecular Endocrinology
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  • 1 Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Correspondence should be addressed to P J D Delhanty: p.delhanty@erasmusmc.nl
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Acylated ghrelin (AG) is a gut-derived peptide with growth hormone secretagogue (GHS), orexigenic and other physiological activities mediated by GHS receptor-1a (GHSR). Ghrelin occurs in unacylated form (UAG) with activities opposing AG, although its mechanism of action is unknown. UAG does not antagonize AG at GHSR, and has biological effects on cells that lack this receptor. Because UAG binds to cells, it has been hypothesized that UAG acts via a cell-surface receptor, although this has not been confirmed. This study aimed to identify cell surface proteins to which UAG binds that could modulate or mediate its biological effects. The MCF7 cell-line was used as a model because UAG induces ERK signaling in these cells in the absence of GHSR. Using ligand–receptor capture and LC-MS/MS we identified specific heparan-sulfate proteoglycans (HSPGs) to which UAG interacts on cell surfaces. In line with this, UAG, as well as AG, bind with high affinity to heparin, and heparin and heparinase treatment suppress, whereas HSPG overexpression increases, UAG binding to MCF7 cell surfaces. Moreover, heparin suppresses the ERK response to UAG. However, conversion of the lysines in UAG to alanine, which prevents its binding to heparin and cell surface HSPGs, does not prevent its activation of ERK. Our data show that the interaction of UAG with HSPGs modulates its biological activity in cells. More broadly, the interaction of UAG and AG with HSPGs could be important for the specificity and potency of their biological action in vivo.

Supplementary Materials

    • Supplemental Figure 1. UAG 6-13 (AZP502) does not bind to cells or heparin. A. FACS plots of MCF7 cells incubated with AZP502 (C-terminal biotin, AZP502-bio; N- and C-terminal biotins, bio-AZP502-bio) at different concentrations . (black, unstained; blue, APC stained; range, 200 nM AZP502; red, 2 μM AZP502; green, 20 μM AZP502). B. FPLC of biotinylated UAG and AZP502 on a heparin-sepharose column. Dot-blots of 2 ul samples from 1 ml fractions. Biotinylated peptides were detected using streptavidin-IrDye800CW fluorescent conjugates and imaged using an Odyssey CLx infra-red scanner. Fractions 1-4 are the flow through, followed by a linear NaCl gradient (0-2 M) from fractions 5-10. AZP502 was eluted in the flow through.

 

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