Identification of a contact domain between echistatin and the integrin alpha(v)beta(3) by photoaffinity cross-linking.
The integrin alpha(v)beta(3) is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the alpha(v)beta(3) receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the beta(3)[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the beta(3)[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the alpha(v)beta(3) receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the alpha(v)beta(3) receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz(2)), located within the flexible C-terminal domain and removed 20 amino acid residues from the R(24)GD(26) triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled (125)I-[Arg(35),Lys(45)(N(epsilon)-pBz(2))]-echistatin photo-cross-links effectively to a site within the beta(3)[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to alpha(IIb)beta(3), the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, beta(3)[99-118] and beta(3)[167-171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin beta(3) subunit differs from the known conformation of I domains.
Scheibler
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Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Mierke
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DF
Bitan
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Rosenblatt
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Chorev
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D019030:Receptors, Vitronectin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C057986:echistatin; C047981:arginyl-glycyl-aspartic acid