Binding and Uptake of RGD-Containing Ligands to Cellular α
v
β
3 Integrins |
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Authors: | Sonya Cressman Ying Sun E Jane Maxwell Ning Fang David D Y Chen Pieter R Cullis |
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Institution: | (1) Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada;(2) Department of Chemistry, University of British Columbia, Vancouver, BC, Canada |
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Abstract: | The cyclic peptide, cRGDfN(me)]V, binds to the α
v
β
3 integrin and can disrupt binding of the integrin to its natural ligands in the extracellular matrix. In this work, the ability
of a water-soluble, fluorescently labeled variant of the RGD-containing peptide (cRGDfK-488) to bind to integrins on human
umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis was characterized. This information was
compared to the binding and uptake properties of an α
v
β
3 integrin-specific monoclonal antibody, LM609X. The specificity of the RGD-containing peptide is assessed by comparison with
control peptide that does not bind to the α
v
β
3 integrin, cRADfK-488. Using a high purity construct, it is shown that the RGD ligand exhibits dissociation constants in the
micromolar range whereas LM609X exhibits dissociation constants in the nanomolar range. However, the RGD ligand showed greater
uptake following incubation at temperatures which permit endocytosis. A 7.4-fold increase in uptake of the RGD peptide was
observed following a 1 h incubation with HUVEC at 37°C (an endocytosis permissive temperature), as compared to that at 4°C
(an endocytosis prohibitive temperature). In contrast, only a 1.9-fold increase in cell-associated fluorescence was observed
for similar incubations with LM609X. Results from fluorescence microscopy supports the notion that the RGD peptide is rapidly
endocytosed at 37°C as compared to LM609X. These results are discussed with regard to previous work indicating that RGD ligands
enter cells by integrin-independent pathways. These studies provide well-controlled measures of how RGD ligands stimulate
endocytosis. This may be of considerable interest for intracellular delivery of ligand-associated drugs in anti-angiogenic
applications. |
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Keywords: | α v β 3 integrins RGD Endocytosis Angiogenesis |
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