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Studies on alpha v beta 3/ligand interactions using a [3H]SK&F-107260 binding assay
A Wong1, S M Hwang, P McDevitt
1Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. angela@spirit.gcrc.upenn.edu
Molecular Pharmacology
|September 1, 1996
Summary
The vitronectin receptor (alpha v beta 3) specifically binds RGD-containing ligands, as shown by a new assay using [3H]SK&F-107260. This receptor differs from the alpha IIb beta 3 receptor, with implications for cell adhesion studies.
Area of Science:
- Integrin biology
- Cell adhesion research
- Biochemistry and pharmacology
Background:
- The vitronectin receptor (integrin alpha v beta 3) is crucial for cell attachment to extracellular matrix proteins containing the arginine-glycine-aspartic acid (RGD) motif.
- Understanding the specific binding properties of alpha v beta 3 is essential for elucidating its role in physiological and pathological processes.
Purpose of the Study:
- To develop and validate a solid-phase microtiter assay for investigating the binding characteristics of purified alpha v beta 3.
- To characterize the ligand specificity and affinity of alpha v beta 3 using a radiolabeled ligand, [3H]SK&F-107260.
- To compare the binding profile of alpha v beta 3 with that of the structurally related alpha IIb beta 3 receptor.
Main Methods:
- Purification of alpha v beta 3 from human platelets, placenta, and chicken osteoclasts.
- Development of a solid-phase microtiter assay using tritiated [3H]SK&F-107260 as the radioligand.
- Saturation binding studies to determine binding affinity (Kd) and maximum binding capacity (Bmax).
- Inhibition assays using RGD- and RGE-containing peptides, echistatin, and alpha IIb beta 3 antagonists.
- Cell adhesion assays using MG63 human osteosarcoma cells and rat osteoclasts on recombinant rat osteopontin.
Main Results:
- Purified alpha v beta 3 from various sources specifically and saturably bound [3H]SK&F-107260.
- Platelet alpha v beta 3 exhibited high-affinity binding with Kd = 1.44 nM and Bmax = 0.20, indicating a single class of binding sites.
- Binding was inhibited by RGD-containing peptides and echistatin but not by RGE peptides or alpha IIb beta 3 antagonists, confirming distinct ligand specificity.
- The binding assay results correlated well with cell adhesion assays, validating the assay's relevance to cell-surface receptor function.
Conclusions:
- The developed [3H]SK&F-107260 binding assay is a valid method for studying purified alpha v beta 3.
- Alpha v beta 3 displays distinct ligand specificity compared to the alpha IIb beta 3 receptor.
- The findings support the utility of this assay for characterizing alpha v beta 3 interactions and evaluating potential antagonists.