title: Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy.
abstract: We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.
authors.affiliation: Klinische Forschergruppe der Frauenklinik der Technischen Universität München (TUM), 81675 München, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Lössner
authors.forename: Horst
authors.initials: H
authors.lastname: Kessler
authors.forename: Georgette
authors.initials: G
authors.lastname: Thumshirn
authors.forename: Claudia
authors.initials: C
authors.lastname: Dahmen
authors.forename: Birgit
authors.initials: B
authors.lastname: Wiltschi
authors.forename: Motomu
authors.initials: M
authors.lastname: Tanaka
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Knoll
authors.forename: Eva-Kathrin
authors.initials: EK
authors.lastname: Sinner
authors.forename: Ute
authors.initials: U
authors.lastname: Reuning
mesh_terms: D000595:Amino Acid Sequence; D001704:Biopolymers / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D001704:Biopolymers; D016023:Integrins; D008024:Ligands; D008051:Lipid Bilayers; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid