Binding of Small Mono- and Oligomeric Integrin Ligands to Membrane-Embedded Integrins Monitored by Surface Plasmon-Enhanced Fluorescence Spectroscopy
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- 作者：Daniela Lö ; ssner ; Horst Kessler ; Georgette Thumshirn ; Claudia Dahmen ; Birgit Wiltschi ; Motomu Tanaka ; Wolfgang Knoll ; Eva-Kathrin Sinner ; and Ute Reuning
- 刊名：Analytical Chemistry
- 出版年：2006
- 出版时间：July 1, 2006
- 年：2006
- 卷：78
- 期：13
- 页码：4524 - 4533
- 全文大小：268K
- 年卷期：v.78,no.13(July 1, 2006)
- ISSN：1520-6882
文摘
We recently developed a binding assay format by incorporating native transmembrane receptors into artificialphospholipid bilayers on biosensor devices for surfaceplasmon resonance spectroscopy. By extending the methodto surface plasmon-enhanced fluorescence spectroscopy(SPFS), sensitive recording of the association of even verysmall ligands is enabled. Herewith, we monitored bindingof synthetic mono- and oligomeric RGD-based peptidesand peptidomimetics to integrins 
rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3 and rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">5, afterhaving confirmed correct orientation and functionality ofmembrane-embedded integrins. We evaluated integrinbinding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimerrevealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic wasalso found to be highly active with a slightly higherselectivity toward rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3. The different compounds werealso evaluated in in vitro cell adhesion tests for theircapacity to interfere with rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3-mediated cell attachmentto vitronectin. We hereby demonstrated that the differentRGD monomers were similarly effective; the RGD dimerand tetramer showed comparable IC50 values, which were,however, significantly higher than those of the monomers.Best cell detachment from vitronectin was achieved by thepeptidomimetic. The novel SPFS-binding assay platformproves to be a suitable, reliable, and sensitive method tomonitor the binding capacity of small ligands to nativetransmembrane receptors, here demonstrated for integrins.
rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3 and rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">5, afterhaving confirmed correct orientation and functionality ofmembrane-embedded integrins. We evaluated integrinbinding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimerrevealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic wasalso found to be highly active with a slightly higherselectivity toward rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3. The different compounds werealso evaluated in in vitro cell adhesion tests for theircapacity to interfere with rs/alpha.gif" BORDER=0>vrs/beta2.gif" BORDER=0 ALIGN="middle">3-mediated cell attachmentto vitronectin. We hereby demonstrated that the differentRGD monomers were similarly effective; the RGD dimerand tetramer showed comparable IC50 values, which were,however, significantly higher than those of the monomers.Best cell detachment from vitronectin was achieved by thepeptidomimetic. The novel SPFS-binding assay platformproves to be a suitable, reliable, and sensitive method tomonitor the binding capacity of small ligands to nativetransmembrane receptors, here demonstrated for integrins.