title: Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a.
abstract: Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl)(3), Lys(fluorescein)(19)]ghrelin(1-19)). Receptor binding affinity of this novel fluorescein-ghrelin(1-18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1-18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1-18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1-18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.
authors: Lawson Health Research Institute, Diabetes and Metabolism and Imaging Programs, 268 Grosvenor St, London, ON, Canada.
authors: Rebecca
authors: R
authors: McGirr
authors: Mark S
authors: MS
authors: McFarland
authors: Jillian
authors: J
authors: McTavish
authors: Leonard G
authors: LG
authors: Luyt
authors: Savita
authors: S
authors: Dhanvantari
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D054439:Ghrelin / Q000031:analogs & derivatives* / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000652:urine; D009206:Myocardium / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000378:metabolism*
chemical_list: D054439:Ghrelin; D054440:Receptors, Ghrelin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016650:Fluorescein-5-isothiocyanate