The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin ␣ IIb ␤ 3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to ␣ IIb ␤ 3 , particularly in the presence of Mn 2؉; IC 50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn 2؉. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in ␣ IIb ␤ 3 . The distance between these sites was estimated to be 6.1 ؎ 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn 2؉. cRGD, but not cHarGD, bound to recombinant ␤ 3 -(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to ␣ IIb ␤ 3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in ␣ IIb ␤ 3 and initiate different functional consequences within the receptor itself and within platelets. ␣ IIb ␤ 3 (GPIIb-IIIa) is a member of integrin family of cell adhesion receptors (1-3) and is the most abundant membrane protein on the platelet surface (4). On nonstimulated platelets, ␣ IIb ␤ 3 is incapable of binding most of its soluble macromolecular ligands (5), but after exposure of the cells to appropriate agonists, the receptor undergoes a conformational change (6) as a consequence of signal transmission from inside the cell to the extracellular domain of the receptor (inside-out signaling (7, 8)) and becomes competent to interact with several plasma protein ligands, including fibrinogen, fibronectin, and von Willebrand factor (9 -11). Fibrinogen inhibits the binding of other two ligands to ␣ IIb ␤ 3 (10 -13), and a common set of monoclonal antibodies (mAbs) 1 to the receptor blocks the interaction of these adhesive ligands with ␣ IIb ␤ 3 (9). Two sets of ligand peptides, ␥ chain peptides, which correspond to the sequence at the carboxyl-terminal sequence of the fibrinogen ␥ chain, and RGD(X) peptides, which correspond to sequences present in all three macromolecular ligands, define the recognition specificity of ␣ IIb ␤ 3 for its macromolecular ligands (reviewed in Ref. 14). Both peptide sets inhib...

The midsegment of the ␤ 3 subunit has been implicated in the ligand and cation binding functions of the ␤ 3 integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, ␤ 3 -(95-373) and ␤ 3 -(95-301), were expressed and found to bind fibrinogen. ␣ IIb ␤ 3 is a typical member of the integrin family of cell adhesion receptors (1), being composed of an ␣ (␣ IIb ) and a ␤ (␤ 3 ) subunit, which associate to form a noncovalent heterodimer. This integrin is the most abundant membrane protein on the platelet surface and serves as a receptor for multiple adhesive proteins including fibrinogen (Fg).1 Two sets of peptides (HHLGGAKQAGDV, corresponding to the sequence at the C terminus of the Fg ␥-chain (2) and RGDX, corresponding to a sequence present in many protein ligands of ␣ IIb ␤ 3 and recognized by many other integrins as well) define the recognition specificity of ␣ IIb ␤ 3 for its macromolecular ligands (reviewed in Ref.3). ␣ V ␤ 3 , which shares the same ␤ 3 subunit as ␣ IIb ␤ 3 , is broadly distributed and binds many but not all of the same ligands as ␣ IIb ␤ 3 , including Fg, von Willebrand factor, and fibronectin (reviewed in Refs. 4 and 5). This integrin also exhibits an RGD recognition specificity but shows a much weaker recognition of Fg ␥-chain peptides (6). Numerous studies have suggested that binding of macromolecular ligands to ␣ IIb ␤ 3 as well as ␣ V ␤ 3 involves multiple contacts in each subunit (7-13). Essential residues for ligand binding to ␣ IIb ␤ 3 reside in two major regions: a midsegment of ␤ 3 , ␤ 3 -(95-400), and the amino-terminal aspect of ␣ IIb , ␣ IIb -(1-334) (11), which contains seven structural repeats (14). The midsegment of the ␤ 3 subunit is highly conserved among integrin ␤ subunits and exhibits some structural and functional features of an I domain. I domains are present in nine integrin ␣ subunits and play major roles in the ligand binding functions of their integrin heterodimers (15)(16)(17)(18)(19)(20). The relationship between the conserved ␤ midsegments and ␣ I domains was proposed based upon similarities in their hydropathy profiles, secondary structural predictions, and mutational analyses (18, 20 -22). A central feature of I domains is a metal ion-dependent adhesion site, a MIDAS motif (15,18,19,23). In MIDAS motifs, three of the five cation coordination sites are provided by a DXSXS sequence and two other coordination sites are provided by oxygenated residues distant in the primary sequence. Mutations of the cation-coordinating residues in a MIDAS motif often cause loss of ligand binding functions of an integrin, and ligand binding sites map in close proximity to MIDAS motifs (18,24,25). The ␤ 3 midsegment does contain a 119 DXSXS sequence, and these residues also have been implicated in the ligand binding functions of ␣ IIb ␤ 3 (24, 26 -28). While there is broad consensus that the midsegment of integrin ␤ subunit contains a functional MIDAS, other structural algorithms have predicted ...

We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.