TY - JOUR
T1 - Unacylated ghrelin binds heparan-sulfate proteoglycans which modulate its function
AU - Delhanty, Patric J.D.
AU - Huisman, Martin
AU - Prins, Karina
AU - Steenbergen, Cobie
AU - Mies, Rosinda
AU - Neggers, Sebastian J.C.M.M.
AU - van der Lely, A. J.
AU - Visser, Jenny A.
PY - 2021/2/1
Y1 - 2021/2/1
N2 - 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.
AB - 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.
KW - cell-surface protein
KW - extracellular-signal-regulated kinase (ERK)
KW - ghrelin
KW - heparan-sulfate proteoglycan (HSPG)
KW - heparin
KW - heparin-binding protein
KW - ligand-receptor capture
KW - peptide hormone
KW - unacylated
UR - http://www.scopus.com/inward/record.url?scp=85101896173&partnerID=8YFLogxK
U2 - 10.1530/JME-20-0286
DO - 10.1530/JME-20-0286
M3 - Article
C2 - 33263557
AN - SCOPUS:85101896173
SN - 1479-6813
VL - 66
SP - 83
EP - 96
JO - Journal of molecular endocrinology
JF - Journal of molecular endocrinology
IS - 2
ER -