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Vol. 58, Issue 5, 1137-1145, November 2000
Laboratoire de Biologie Cellulaire de l'Hypertension,
Institut de Recherches Cliniques de Montréal and Université
de Montréal, Montréal, Quebec, Canada
This study shows that disintegrins, echistatin as a model, can be used
as a radiolabeled probe to simultaneously detect the presence of
individual RGD-dependent integrins on cardiac fibroblasts. Binding of
125I-echistatin to fibroblasts was proportional to cell
number, time dependent, reversible, saturable, specific, and membrane
bound. SDS-polyacrylamide gel electrophoresis and autoradiograms
revealed that 125I-echistatin was associated with three
radioactive protein bands of 180, 210, and 220 kDa that were identified
by RGD affinity chromatography, immunoblotting, and
immunoneutralization as 
v
3, 31/51/v1,
and 81 heterodimeric integrins,
respectively. These results suggest that echistatin binds to
RGD-dependent integrins, forming SDS-stable complexes in the absence of
chemical cross-linkers, reducing conditions and heating. As assessed by
radioligand-binding filtration, disintegrins displayed binding
characteristics with an IC50 ranging from 0.044 to 1.1 nM,
but with slope factors lower than 1, indicating the presence of several
binding sites. Resolved by SDS-polyacrylamide gel electrophoresis to
reveal echistatin-integrin complexes, disintegrins and RGD peptides
displayed different binding affinities to individual RGD-dependent
integrins present on cardiac fibroblasts. Elegantin and flavostatin
demonstrated the highest affinity toward integrins, whereas flavoridin
and acPenRGDC had a greater specificity toward
v3-integrin. In summary, echistatin forms
SDS-stable complexes with RGD-dependent integrins. This model offers a
novel way to visualize RGD-dependent integrins, to investigate their
activation state, and to determine the integrin specificity of RGD peptides.
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