Sodium dodecyl sulfate-stable complexes of echistatin and RGD-dependent integrins: a novel approach to study integrins.
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 (125)I-echistatin to fibroblasts was proportional to cell number, time dependent, reversible, saturable, specific, and membrane bound. SDS-polyacrylamide gel electrophoresis and autoradiograms revealed that (125)I-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 alpha(v)beta(3), alpha(3)beta(1)/alpha(5)beta(1)/alpha(v)beta(1), and alpha(8)beta(1) 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 IC(50) 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 alpha(v)beta(3)-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.
Thibault
G
G
Laboratoire de Biologie Cellulaire de l'Hypertension, Institut de Recherches Cliniques de Montréal and Université de Montréal, Montréal, Quebec, Canada. thibaug@ircm.qc.ca
D000818:Animals; D001665:Binding Sites; D019483:Disintegrins / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D016023:Integrins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009206:Myocardium / Q000166:cytology; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
D019483:Disintegrins; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D010455:Peptides; C057986:echistatin; D012967:Sodium Dodecyl Sulfate; C047981:arginyl-glycyl-aspartic acid