RGD-containing Peptides Inhibit Fibrinogen Binding to Platelet αIIbβ3 by Inducing an Allosteric Change in the Amino-terminal Portion of αIIb
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m011511200
ISSN1083-351X
AutoresRamesh B. Basani, Giovanna D’Andrea, Neal Mitra, Gaston Vilaire, Mark Richberg, M. Anna Kowalska, Joel Bennett, Mortimer Poncz,
Tópico(s)Cell Adhesion Molecules Research
ResumoTo determine the molecular basis for the insensitivity of rat αIIbβ3 to inhibition by RGD-containing peptides, hybrids of human and rat αIIbβ3 and chimeras of αIIbβ3 in which αIIb was composed of portions of human and rat αIIb were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of αIIbβ3 was measured. These measurements indicated that sequences regulating the sensitivity of αIIbβ3 to RGDS are located in the seven amino-terminal repeats of αIIb. Moreover, replacing the first three or four (but not the first two) repeats of rat αIIb with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human αIIb with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing αIIbβ3 regardless whether the αIIb in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of αIIbβ3 to RGD-containing peptides are located in the third and fourth amino-terminal repeats of αIIb. Because RGDS binds to both human and rat αIIbβ3, the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding. To determine the molecular basis for the insensitivity of rat αIIbβ3 to inhibition by RGD-containing peptides, hybrids of human and rat αIIbβ3 and chimeras of αIIbβ3 in which αIIb was composed of portions of human and rat αIIb were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of αIIbβ3 was measured. These measurements indicated that sequences regulating the sensitivity of αIIbβ3 to RGDS are located in the seven amino-terminal repeats of αIIb. Moreover, replacing the first three or four (but not the first two) repeats of rat αIIb with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human αIIb with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing αIIbβ3 regardless whether the αIIb in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of αIIbβ3 to RGD-containing peptides are located in the third and fourth amino-terminal repeats of αIIb. Because RGDS binds to both human and rat αIIbβ3, the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding. Ligand binding to integrins initiates intracellular signals that are crucial for cellular growth and differentiation (1Hynes R.O. Cell.. 1987; 48: 549-554Google Scholar). Conversely, many cells regulate the ability of their integrins to recognize ligands. The prototypic example of integrin regulation is the platelet integrin αIIbβ3 (2Bennett J.S. Trends Cardiovasc. Med... 1996; 16: 31-36Google Scholar). In unstimulated platelets, αIIbβ3 is inactive; but following platelet stimulation by agonists such as ADP and thrombin, αIIbβ3 assumes a conformation in which it is able to bind macromolecular ligands such as fibrinogen and von Willebrand factor. Because ligand binding to αIIbβ3 is a prerequisite for platelet aggregation, regulating the affinity of αIIbβ3 for ligands assures that only stimulated platelets aggregate.The major ligand for αIIbβ3 in plasma is fibrinogen. Three portions of the fibrinogen molecule (the carboxyl terminus of the fibrinogen γ-chain (3Kloczewiak M. Timmons S. Hawiger J. Biochem. Biophys. Res. Commun... 1982; 107: 181-187Google Scholar) and two Arg-Gly-Asp (RGD) motifs located in the fibrinogen α-chain (4Gartner T.K. Bennett J.S. J. Biol. Chem... 1985; 260: 11891-11894Google Scholar)) have been proposed to be sites that mediate fibrinogen binding to αIIbβ3. However, ultrastructural examination of fibrinogen bound to αIIbβ3(5Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem... 1992; 267: 16637-16643Google Scholar) and measurements of fibrinogen binding to αIIbβ3 using fibrinogens containing mutated RGD or γ-chain sequences (6Farrell D.H. Thiagarajan P. J. Biol. Chem... 1994; 269: 226-231Google Scholar) indicate that it is the γ-chain sequences that mediate fibrinogen binding. Nonetheless, RGD-containing disintegrins and synthetic compounds based on the RGD motif are effective αIIbβ3 antagonists (7Lefkovits J. Plow E. Topol E. N. Engl. J. Med... 1995; 332: 1553-1559Google Scholar), implying that they either directly or indirectly affect the γ-chain-binding site when they bind to αIIbβ3.Ligands appear to bind to αIIbβ3 by interacting with the amino-terminal portion of β3 (8Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem... 2000; 275: 7795-7802Google Scholar), although the specific residues involved are not known. A region of β3 encoded by the fourth and fifth exons of the β3 gene that encompasses amino acids 95–223 has been implicated in ligand binding (9Lin E.C.K. Ratnikov B.I. Tsai P.M. Carron C.P. Myers D.M. Barbas III, C.F. Smith J.W. J. Biol. Chem... 1997; 272: 23912-23920Google Scholar). Moreover, chemical cross-linking experiments have suggested that RGD-containing peptides bind to β3 in the vicinity of amino acids 109–171 (10D'Souza S.E. Ginsberg M.H. Burke T.A. Lam S.C.-T. Plow E.F. Science.. 1988; 242: 91-93Google Scholar). It is noteworthy that this region of β3 contains an array of oxygenated residues whose three-dimensional structure may resemble that of the ligand-binding I domains that are present in several integrin α-subunits (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar). In addition, overlapping peptides corresponding to β3 amino acids 211–222 inhibit fibrinogen binding to purified αIIbβ3, suggesting that this stretch of residues represents a portion of the fibrinogen-binding site (12Charo I.F. Nannizzi L. Phillips D.R. Hsu M.A. Scarborough R.M. J. Biol. Chem... 1991; 266: 1415-1421Google Scholar, 13Steiner B. Trzeciak A. Pfenninger G. Kouns W. J. Biol. Chem... 1993; 268: 6870-6873Google Scholar). There is also evidence that more distal portions of β3 may be involved in fibrinogen binding because a naturally occurring Leu262 → Pro mutation prevents αIIbβ3 binding to immobilized fibrinogen (14Ward C.M. Kestin A.S. Newman P.J. Blood.. 2000; 96: 161-169Google Scholar).Ligand binding to αIIbβ3 also appears to involve the amino-terminal third of αIIb (15Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem... 1996; 271: 2033-2039Google Scholar). Chemical cross-linking experiments suggest that the carboxyl terminus of the fibrinogen γ-chain binds to αIIb in the vicinity of amino acids 294–314 (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar), a suggestion supported by the ability of a peptide corresponding to αIIb residues 300–312 to inhibit platelet adhesion to fibrinogen (17Taylor D.B. Gartner T.K. J. Biol. Chem... 1992; 267: 11729-11733Google Scholar). In addition, there are a number of reports of naturally occurring and laboratory-induced mutations involving amino acids located between αIIbresidues 183 and 224 that impair αIIbβ3function, suggesting that this portion of αIIb binds to ligands as well (18Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem... 1996; 271: 18610-18615Google Scholar, 19Grimaldi C.M. Chen F. Wu C. Weiss H.J. Coller B.S. French D.L. Blood.. 1998; 91: 1562-1571Google Scholar, 20Tozer E.C. Baker E.K. Ginsberg M.H. Loftus J.C. Blood.. 1999; 93: 918-924Google Scholar, 21Basani R.B. French D.L. Vilaire G. Brown D.L. Chen F. Coller B.S. Derrick J.M. Gartner T.K. Bennett J.S. Poncz M. Blood.. 2000; 95: 180-188Google Scholar).Although fibrinogen binding to αIIbβ3 on the platelets of all mammalian species is required for platelet aggregation, there are substantial differences in the ability of RGD-containing peptides to inhibit the process. For example, fibrinogen binding to rabbit and rat platelets is relatively insensitive to inhibition by RGD-containing peptides (22Harfenist E.J. Packham M.A. Mustard J.F. Blood.. 1988; 71: 132-136Google Scholar, 23Jennings L.K. White M.M. Mandrell T.D. Thromb. Haemostasis.. 1995; 74: 1551-1556Google Scholar). To gain an understanding of the molecular basis for the insensitivity of rat αIIbβ3 to RGD-containing peptides, we measured the effect of the tetrapeptide Arg-Gly-Asp-Ser (RGDS) on fibrinogen binding to chimeric αIIbβ3molecules composed of portions of the rat and human proteins. We found that the sequences determining the sensitivity or resistance of αIIbβ3 to inhibition by RGDS are located in the third and fourth repeats of the amino-terminal portion of αIIb. Moreover, because we also found that RGDS bound to αIIbβ3 regardless of whether the heterodimer contained human or rat subunits, our results imply that RGDS impairs fibrinogen binding to αIIbβ3by inducing an allosteric change in the third and fourth repeats of αIIb. They also suggest that a conformational change in these repeats may be required for the fibrinogen binding to αIIbβ3 that occurs on agonist-stimulated platelets.DISCUSSIONAlthough fibrinogen appears to bind to αIIbβ3 exclusively via sequences located at the carboxyl-terminal end of the fibrinogen γ-chain (5Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem... 1992; 267: 16637-16643Google Scholar, 6Farrell D.H. Thiagarajan P. J. Biol. Chem... 1994; 269: 226-231Google Scholar), peptides containing an RGD motif are competitive inhibitors of fibrinogen binding to αIIbβ3 (4Gartner T.K. Bennett J.S. J. Biol. Chem... 1985; 260: 11891-11894Google Scholar). Moreover, despite chemical cross-linking experiments suggesting that the γ-chain and RGD-containing peptides bind to different subunits of the αIIbβ3 heterodimer (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar), competitive binding measurements indicate that the peptides bind to αIIbβ3 in a mutually exclusive manner (39Bennett J.S. Shattil S.J. Power J.W. Gartner T.K. J. Biol. Chem... 1988; 263: 12948-12953Google Scholar), implying either that the peptides bind to same site or that the binding sites interact allosterically. Hu et al. (40Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem... 1999; 274: 4633-4639Google Scholar), using plasmon resonance spectroscopy to study the effect of RGD ligands on fibrinogen binding to αIIbβ3, concluded that fibrinogen and RGD ligands bind to separate sites on αIIbβ3, but suggested that there is an allosteric relationship between the two. Using chimeras of RGD-insensitive rat αIIbβ3 and RGD-sensitive human αIIbβ3, we found that sensitivity to the inhibitory effects of the tetrapeptide RGDS was determined by the origin of the third and fourth amino-terminal repeats of αIIb. We also found little difference in the affinity of αIIbβ3 on human and rat platelets for fibrinogen. Thus, our data suggest that rather than directly affecting fibrinogen binding, species differences in the third and fourth αIIb repeats affect an allosteric change that regulates fibrinogen binding to αIIbβ3.Ligand binding to αIIbβ3 is thought to involve regions located in the amino-terminal portions of both αIIb and β3 (8Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem... 2000; 275: 7795-7802Google Scholar), although much of this evidence is indirect. The β3 region encompasses amino acids 95–223 (9Lin E.C.K. Ratnikov B.I. Tsai P.M. Carron C.P. Myers D.M. Barbas III, C.F. Smith J.W. J. Biol. Chem... 1997; 272: 23912-23920Google Scholar) and includes the RGD-cross-linking site located in the vicinity of amino acids 109–171 (38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar) as well as an array of oxygenated residues whose fold may resemble that of the ligand-bindingmetal ion-dependentadhesion sites (MIDAS) present in integrin I domains (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar). It is noteworthy that the deleterious effect of an Arg214 → Trp mutation, located in the midst of this sequence, can be reversed by exposing αIIbβ3 to DTT, suggesting that the presence of Trp at residue 214 does not prevent fibrinogen binding to αIIbβ3 directly, but rather obscures the fibrinogen-binding site (41Kouns W. Steiner B. Kunicki T. Moog S. Jutzi J. Jennings L. Cazenave J. Lanza F. Blood.. 1994; 84: 1015-1108Google Scholar).It is also noteworthy that the location of the binding site for RGD-containing peptides in integrins is uncertain, and there is evidence for binding sites in both α- and β-subunits. For example, proteins corresponding to the fourth through seventh amino-terminal repeats of α5 and αIIb bind to fibronectin III fragment-(8–10) and to fibrinogen, respectively, in an RGD- dependent manner (42Baneres J.L. Roquet F. Green M. LeCalvez H. Parello J. J. Biol. Chem... 1998; 273: 24744-24753Google Scholar, 43Gulino D. Boudignon C. Zhang L. Concord E. Rabiet M.-J. Marguerie G. J. Biol. Chem... 1992; 267: 1001-1007Google Scholar). Conversely, experiments using chemical and photoaffinity cross-linking, site-directed mutagenesis, synthetic integrin and RGD-containing peptides, and mAbs have identified regions in the amino-terminal portion of β1- and β3-subunits that recognize the RGD motif (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar,44Bitan G. Scheibler L. Greenberg Z. Rosenblatt M. Chorev M. Biochemistry.. 1999; 38: 3414-3420Google Scholar, 45Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem... 1997; 272: 17283-17292Google Scholar, 46Humphries J.D. Askari J.A. Zhang X.P. Takada Y. Humphries M.J. Mould A.P. J. Biol. Chem... 2000; 275: 20337-20345Google Scholar). Based on these observations, one possible explanation for our results is simply that RGDS does not bind to either rat αIIb or rat β3. However, we found that first, the sensitivity of αIIbβ3 composed of human subunits or of a human α-subunit and rat β-subunit to RGDS was equivalent, and second, binding of mAb 10-758 to human β3 was induced by RGDS to an equal extent regardless of whether αIIb was human, rat, or a human-rat chimera. Thus, our data imply that RGDS binds to both human and rat αIIbβ3 and that differences in its inhibitory potency are due to differences in allosteric events that follow RGDS binding.The portion of αIIb implicated in ligand binding has also been localized to the amino-terminal third of the molecule (15Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem... 1996; 271: 2033-2039Google Scholar) and includes the fibrinogen γ-chain peptide-cross-linking site at amino acids 294–314 (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar). In addition, a number of naturally occurring and laboratory-induced mutations involving amino acids 145, 183, 184, 189, 190, 191, 193, and 224 have been described that impair αIIbβ3 function, suggesting that these residues interact with αIIbβ3 ligands (18Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem... 1996; 271: 18610-18615Google Scholar, 19Grimaldi C.M. Chen F. Wu C. Weiss H.J. Coller B.S. French D.L. Blood.. 1998; 91: 1562-1571Google Scholar, 20Tozer E.C. Baker E.K. Ginsberg M.H. Loftus J.C. Blood.. 1999; 93: 918-924Google Scholar). Of note, residues 183–224 are located in the third αIIb repeat (25Poncz M. Eisman R. Heidenreich R. Silver S.M. Vilaire G. Surrey S. Schwartz E. Bennett J.S. J. Biol. Chem... 1987; 262: 8476-8482Google Scholar). Because our data suggest that the third repeat is involved in the allosteric regulation of fibrinogen binding to αIIbβ3, it is possible that mutation of the residues listed above interferes with this allosteric change, rather than directly perturbing the fibrinogen-binding site.The tertiary structure of integrins has yet to be determined. Based on computer modeling, Springer (31Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 65-72Google Scholar) proposed that the amino-terminal portion of integrin α-subunits folds into a seven-bladed β-propeller configuration, with each of the blades corresponding to a β-sheet formed from four anti-parallel β-strands located within each of the amino-terminal repeats. Loops connecting the β-strands would be located on either the upper or low surface of the proposed propeller such that residues in three loops in human αIIbbetween Arg147 and Tyr166, Val182and Leu195, and His215 and Gly233, connecting portions of the third and fourth propeller blades, would be juxtaposed in one quadrant of the upper surface of the propeller (21Basani R.B. French D.L. Vilaire G. Brown D.L. Chen F. Coller B.S. Derrick J.M. Gartner T.K. Bennett J.S. Poncz M. Blood.. 2000; 95: 180-188Google Scholar). Comparison of the amino acid sequence of the loops in human αIIb with that of the analogous portions of rat αIIb (47Thornton M.A. Poncz M. Blood.. 1999; 94: 3947-3950Google Scholar) indicates that the putative second loop is fully conserved, whereas the first and third loops would be only 50% homologous. Thus, it is possible that amino acid sequence differences between human and rat αIIb in the putative first and third loops could be responsible for the differences in sensitivity of human and rat αIIbβ3 to RGD-containing peptides.Alterations in the tertiary and/or quaternary structure of integrins regulate their affinity, and possibly their avidity, for ligands. Recent nuclear magnetic resonance spectroscopic and x-ray crystallographic studies of the I domain of αL emphasize the importance of changes in the conformation of the α-subunit amino terminus in integrin function (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar, 49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). I domains are present in nine integrin α-subunits, where they are inserted between the second and third amino-terminal repeats (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). In αL and αM, ligands such as ICAM-1–3 (intercellular adhesionmolecule) bind to a divalent cation-containing MIDAS motif on the upper I domain surface (50Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure.. 1995; 3: 1333-1340Google Scholar, 51Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 10277-10281Google Scholar, 52Liddington R. Bankston L. Structure.. 1998; 6: 937-938Google Scholar, 53Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 2215-2220Google Scholar). In the I domain of αL, residues distal to the MIDAS motif, lining a cleft formed by the seventh α-helix and the central β-sheet, regulate ligand binding to αLβ2 allosterically (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar) and constitute the binding site for the αLβ2 inhibitor lovastatin (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar). In addition, mutations in the amino- and carboxyl-terminal linker sequences that connect the I domain to the rest of αLeither activate or inactivate I domain function (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar), implying that the changes in I domain conformation that regulate its function are transmitted from the amino-terminal portion of αL to the I domain via these sequences. In the case of αIIbβ3, agonist-induced changes in tertiary structure are essential for its function (2Bennett J.S. Trends Cardiovasc. Med... 1996; 16: 31-36Google Scholar). Our results indicate that an allosteric change in the third and fourth amino-terminal repeats of αIIb, a portion of αIIb located immediately downstream from the I domain insertion site in I domain-containing integrins, regulates ligand binding to αIIbβ3. Thus, by extrapolation, our data suggest that allosteric changes involving the third and fourth α-subunit repeats may be a general mechanism by which ligand binding to integrins is regulated. Ligand binding to integrins initiates intracellular signals that are crucial for cellular growth and differentiation (1Hynes R.O. Cell.. 1987; 48: 549-554Google Scholar). Conversely, many cells regulate the ability of their integrins to recognize ligands. The prototypic example of integrin regulation is the platelet integrin αIIbβ3 (2Bennett J.S. Trends Cardiovasc. Med... 1996; 16: 31-36Google Scholar). In unstimulated platelets, αIIbβ3 is inactive; but following platelet stimulation by agonists such as ADP and thrombin, αIIbβ3 assumes a conformation in which it is able to bind macromolecular ligands such as fibrinogen and von Willebrand factor. Because ligand binding to αIIbβ3 is a prerequisite for platelet aggregation, regulating the affinity of αIIbβ3 for ligands assures that only stimulated platelets aggregate. The major ligand for αIIbβ3 in plasma is fibrinogen. Three portions of the fibrinogen molecule (the carboxyl terminus of the fibrinogen γ-chain (3Kloczewiak M. Timmons S. Hawiger J. Biochem. Biophys. Res. Commun... 1982; 107: 181-187Google Scholar) and two Arg-Gly-Asp (RGD) motifs located in the fibrinogen α-chain (4Gartner T.K. Bennett J.S. J. Biol. Chem... 1985; 260: 11891-11894Google Scholar)) have been proposed to be sites that mediate fibrinogen binding to αIIbβ3. However, ultrastructural examination of fibrinogen bound to αIIbβ3(5Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem... 1992; 267: 16637-16643Google Scholar) and measurements of fibrinogen binding to αIIbβ3 using fibrinogens containing mutated RGD or γ-chain sequences (6Farrell D.H. Thiagarajan P. J. Biol. Chem... 1994; 269: 226-231Google Scholar) indicate that it is the γ-chain sequences that mediate fibrinogen binding. Nonetheless, RGD-containing disintegrins and synthetic compounds based on the RGD motif are effective αIIbβ3 antagonists (7Lefkovits J. Plow E. Topol E. N. Engl. J. Med... 1995; 332: 1553-1559Google Scholar), implying that they either directly or indirectly affect the γ-chain-binding site when they bind to αIIbβ3. Ligands appear to bind to αIIbβ3 by interacting with the amino-terminal portion of β3 (8Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem... 2000; 275: 7795-7802Google Scholar), although the specific residues involved are not known. A region of β3 encoded by the fourth and fifth exons of the β3 gene that encompasses amino acids 95–223 has been implicated in ligand binding (9Lin E.C.K. Ratnikov B.I. Tsai P.M. Carron C.P. Myers D.M. Barbas III, C.F. Smith J.W. J. Biol. Chem... 1997; 272: 23912-23920Google Scholar). Moreover, chemical cross-linking experiments have suggested that RGD-containing peptides bind to β3 in the vicinity of amino acids 109–171 (10D'Souza S.E. Ginsberg M.H. Burke T.A. Lam S.C.-T. Plow E.F. Science.. 1988; 242: 91-93Google Scholar). It is noteworthy that this region of β3 contains an array of oxygenated residues whose three-dimensional structure may resemble that of the ligand-binding I domains that are present in several integrin α-subunits (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar). In addition, overlapping peptides corresponding to β3 amino acids 211–222 inhibit fibrinogen binding to purified αIIbβ3, suggesting that this stretch of residues represents a portion of the fibrinogen-binding site (12Charo I.F. Nannizzi L. Phillips D.R. Hsu M.A. Scarborough R.M. J. Biol. Chem... 1991; 266: 1415-1421Google Scholar, 13Steiner B. Trzeciak A. Pfenninger G. Kouns W. J. Biol. Chem... 1993; 268: 6870-6873Google Scholar). There is also evidence that more distal portions of β3 may be involved in fibrinogen binding because a naturally occurring Leu262 → Pro mutation prevents αIIbβ3 binding to immobilized fibrinogen (14Ward C.M. Kestin A.S. Newman P.J. Blood.. 2000; 96: 161-169Google Scholar). Ligand binding to αIIbβ3 also appears to involve the amino-terminal third of αIIb (15Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem... 1996; 271: 2033-2039Google Scholar). Chemical cross-linking experiments suggest that the carboxyl terminus of the fibrinogen γ-chain binds to αIIb in the vicinity of amino acids 294–314 (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar), a suggestion supported by the ability of a peptide corresponding to αIIb residues 300–312 to inhibit platelet adhesion to fibrinogen (17Taylor D.B. Gartner T.K. J. Biol. Chem... 1992; 267: 11729-11733Google Scholar). In addition, there are a number of reports of naturally occurring and laboratory-induced mutations involving amino acids located between αIIbresidues 183 and 224 that impair αIIbβ3function, suggesting that this portion of αIIb binds to ligands as well (18Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem... 1996; 271: 18610-18615Google Scholar, 19Grimaldi C.M. Chen F. Wu C. Weiss H.J. Coller B.S. French D.L. Blood.. 1998; 91: 1562-1571Google Scholar, 20Tozer E.C. Baker E.K. Ginsberg M.H. Loftus J.C. Blood.. 1999; 93: 918-924Google Scholar, 21Basani R.B. French D.L. Vilaire G. Brown D.L. Chen F. Coller B.S. Derrick J.M. Gartner T.K. Bennett J.S. Poncz M. Blood.. 2000; 95: 180-188Google Scholar). Although fibrinogen binding to αIIbβ3 on the platelets of all mammalian species is required for platelet aggregation, there are substantial differences in the ability of RGD-containing peptides to inhibit the process. For example, fibrinogen binding to rabbit and rat platelets is relatively insensitive to inhibition by RGD-containing peptides (22Harfenist E.J. Packham M.A. Mustard J.F. Blood.. 1988; 71: 132-136Google Scholar, 23Jennings L.K. White M.M. Mandrell T.D. Thromb. Haemostasis.. 1995; 74: 1551-1556Google Scholar). To gain an understanding of the molecular basis for the insensitivity of rat αIIbβ3 to RGD-containing peptides, we measured the effect of the tetrapeptide Arg-Gly-Asp-Ser (RGDS) on fibrinogen binding to chimeric αIIbβ3molecules composed of portions of the rat and human proteins. We found that the sequences determining the sensitivity or resistance of αIIbβ3 to inhibition by RGDS are located in the third and fourth repeats of the amino-terminal portion of αIIb. Moreover, because we also found that RGDS bound to αIIbβ3 regardless of whether the heterodimer contained human or rat subunits, our results imply that RGDS impairs fibrinogen binding to αIIbβ3by inducing an allosteric change in the third and fourth repeats of αIIb. They also suggest that a conformational change in these repeats may be required for the fibrinogen binding to αIIbβ3 that occurs on agonist-stimulated platelets. DISCUSSIONAlthough fibrinogen appears to bind to αIIbβ3 exclusively via sequences located at the carboxyl-terminal end of the fibrinogen γ-chain (5Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem... 1992; 267: 16637-16643Google Scholar, 6Farrell D.H. Thiagarajan P. J. Biol. Chem... 1994; 269: 226-231Google Scholar), peptides containing an RGD motif are competitive inhibitors of fibrinogen binding to αIIbβ3 (4Gartner T.K. Bennett J.S. J. Biol. Chem... 1985; 260: 11891-11894Google Scholar). Moreover, despite chemical cross-linking experiments suggesting that the γ-chain and RGD-containing peptides bind to different subunits of the αIIbβ3 heterodimer (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar), competitive binding measurements indicate that the peptides bind to αIIbβ3 in a mutually exclusive manner (39Bennett J.S. Shattil S.J. Power J.W. Gartner T.K. J. Biol. Chem... 1988; 263: 12948-12953Google Scholar), implying either that the peptides bind to same site or that the binding sites interact allosterically. Hu et al. (40Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem... 1999; 274: 4633-4639Google Scholar), using plasmon resonance spectroscopy to study the effect of RGD ligands on fibrinogen binding to αIIbβ3, concluded that fibrinogen and RGD ligands bind to separate sites on αIIbβ3, but suggested that there is an allosteric relationship between the two. Using chimeras of RGD-insensitive rat αIIbβ3 and RGD-sensitive human αIIbβ3, we found that sensitivity to the inhibitory effects of the tetrapeptide RGDS was determined by the origin of the third and fourth amino-terminal repeats of αIIb. We also found little difference in the affinity of αIIbβ3 on human and rat platelets for fibrinogen. Thus, our data suggest that rather than directly affecting fibrinogen binding, species differences in the third and fourth αIIb repeats affect an allosteric change that regulates fibrinogen binding to αIIbβ3.Ligand binding to αIIbβ3 is thought to involve regions located in the amino-terminal portions of both αIIb and β3 (8Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem... 2000; 275: 7795-7802Google Scholar), although much of this evidence is indirect. The β3 region encompasses amino acids 95–223 (9Lin E.C.K. Ratnikov B.I. Tsai P.M. Carron C.P. Myers D.M. Barbas III, C.F. Smith J.W. J. Biol. Chem... 1997; 272: 23912-23920Google Scholar) and includes the RGD-cross-linking site located in the vicinity of amino acids 109–171 (38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar) as well as an array of oxygenated residues whose fold may resemble that of the ligand-bindingmetal ion-dependentadhesion sites (MIDAS) present in integrin I domains (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar). It is noteworthy that the deleterious effect of an Arg214 → Trp mutation, located in the midst of this sequence, can be reversed by exposing αIIbβ3 to DTT, suggesting that the presence of Trp at residue 214 does not prevent fibrinogen binding to αIIbβ3 directly, but rather obscures the fibrinogen-binding site (41Kouns W. Steiner B. Kunicki T. Moog S. Jutzi J. Jennings L. Cazenave J. Lanza F. Blood.. 1994; 84: 1015-1108Google Scholar).It is also noteworthy that the location of the binding site for RGD-containing peptides in integrins is uncertain, and there is evidence for binding sites in both α- and β-subunits. For example, proteins corresponding to the fourth through seventh amino-terminal repeats of α5 and αIIb bind to fibronectin III fragment-(8–10) and to fibrinogen, respectively, in an RGD- dependent manner (42Baneres J.L. Roquet F. Green M. LeCalvez H. Parello J. J. Biol. Chem... 1998; 273: 24744-24753Google Scholar, 43Gulino D. Boudignon C. Zhang L. Concord E. Rabiet M.-J. Marguerie G. J. Biol. Chem... 1992; 267: 1001-1007Google Scholar). Conversely, experiments using chemical and photoaffinity cross-linking, site-directed mutagenesis, synthetic integrin and RGD-containing peptides, and mAbs have identified regions in the amino-terminal portion of β1- and β3-subunits that recognize the RGD motif (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar,44Bitan G. Scheibler L. Greenberg Z. Rosenblatt M. Chorev M. Biochemistry.. 1999; 38: 3414-3420Google Scholar, 45Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem... 1997; 272: 17283-17292Google Scholar, 46Humphries J.D. Askari J.A. Zhang X.P. Takada Y. Humphries M.J. Mould A.P. J. Biol. Chem... 2000; 275: 20337-20345Google Scholar). Based on these observations, one possible explanation for our results is simply that RGDS does not bind to either rat αIIb or rat β3. However, we found that first, the sensitivity of αIIbβ3 composed of human subunits or of a human α-subunit and rat β-subunit to RGDS was equivalent, and second, binding of mAb 10-758 to human β3 was induced by RGDS to an equal extent regardless of whether αIIb was human, rat, or a human-rat chimera. Thus, our data imply that RGDS binds to both human and rat αIIbβ3 and that differences in its inhibitory potency are due to differences in allosteric events that follow RGDS binding.The portion of αIIb implicated in ligand binding has also been localized to the amino-terminal third of the molecule (15Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem... 1996; 271: 2033-2039Google Scholar) and includes the fibrinogen γ-chain peptide-cross-linking site at amino acids 294–314 (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar). In addition, a number of naturally occurring and laboratory-induced mutations involving amino acids 145, 183, 184, 189, 190, 191, 193, and 224 have been described that impair αIIbβ3 function, suggesting that these residues interact with αIIbβ3 ligands (18Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem... 1996; 271: 18610-18615Google Scholar, 19Grimaldi C.M. Chen F. Wu C. Weiss H.J. Coller B.S. French D.L. Blood.. 1998; 91: 1562-1571Google Scholar, 20Tozer E.C. Baker E.K. Ginsberg M.H. Loftus J.C. Blood.. 1999; 93: 918-924Google Scholar). Of note, residues 183–224 are located in the third αIIb repeat (25Poncz M. Eisman R. Heidenreich R. Silver S.M. Vilaire G. Surrey S. Schwartz E. Bennett J.S. J. Biol. Chem... 1987; 262: 8476-8482Google Scholar). Because our data suggest that the third repeat is involved in the allosteric regulation of fibrinogen binding to αIIbβ3, it is possible that mutation of the residues listed above interferes with this allosteric change, rather than directly perturbing the fibrinogen-binding site.The tertiary structure of integrins has yet to be determined. Based on computer modeling, Springer (31Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 65-72Google Scholar) proposed that the amino-terminal portion of integrin α-subunits folds into a seven-bladed β-propeller configuration, with each of the blades corresponding to a β-sheet formed from four anti-parallel β-strands located within each of the amino-terminal repeats. Loops connecting the β-strands would be located on either the upper or low surface of the proposed propeller such that residues in three loops in human αIIbbetween Arg147 and Tyr166, Val182and Leu195, and His215 and Gly233, connecting portions of the third and fourth propeller blades, would be juxtaposed in one quadrant of the upper surface of the propeller (21Basani R.B. French D.L. Vilaire G. Brown D.L. Chen F. Coller B.S. Derrick J.M. Gartner T.K. Bennett J.S. Poncz M. Blood.. 2000; 95: 180-188Google Scholar). Comparison of the amino acid sequence of the loops in human αIIb with that of the analogous portions of rat αIIb (47Thornton M.A. Poncz M. Blood.. 1999; 94: 3947-3950Google Scholar) indicates that the putative second loop is fully conserved, whereas the first and third loops would be only 50% homologous. Thus, it is possible that amino acid sequence differences between human and rat αIIb in the putative first and third loops could be responsible for the differences in sensitivity of human and rat αIIbβ3 to RGD-containing peptides.Alterations in the tertiary and/or quaternary structure of integrins regulate their affinity, and possibly their avidity, for ligands. Recent nuclear magnetic resonance spectroscopic and x-ray crystallographic studies of the I domain of αL emphasize the importance of changes in the conformation of the α-subunit amino terminus in integrin function (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar, 49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). I domains are present in nine integrin α-subunits, where they are inserted between the second and third amino-terminal repeats (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). In αL and αM, ligands such as ICAM-1–3 (intercellular adhesionmolecule) bind to a divalent cation-containing MIDAS motif on the upper I domain surface (50Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure.. 1995; 3: 1333-1340Google Scholar, 51Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 10277-10281Google Scholar, 52Liddington R. Bankston L. Structure.. 1998; 6: 937-938Google Scholar, 53Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 2215-2220Google Scholar). In the I domain of αL, residues distal to the MIDAS motif, lining a cleft formed by the seventh α-helix and the central β-sheet, regulate ligand binding to αLβ2 allosterically (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar) and constitute the binding site for the αLβ2 inhibitor lovastatin (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar). In addition, mutations in the amino- and carboxyl-terminal linker sequences that connect the I domain to the rest of αLeither activate or inactivate I domain function (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar), implying that the changes in I domain conformation that regulate its function are transmitted from the amino-terminal portion of αL to the I domain via these sequences. In the case of αIIbβ3, agonist-induced changes in tertiary structure are essential for its function (2Bennett J.S. Trends Cardiovasc. Med... 1996; 16: 31-36Google Scholar). Our results indicate that an allosteric change in the third and fourth amino-terminal repeats of αIIb, a portion of αIIb located immediately downstream from the I domain insertion site in I domain-containing integrins, regulates ligand binding to αIIbβ3. Thus, by extrapolation, our data suggest that allosteric changes involving the third and fourth α-subunit repeats may be a general mechanism by which ligand binding to integrins is regulated. Although fibrinogen appears to bind to αIIbβ3 exclusively via sequences located at the carboxyl-terminal end of the fibrinogen γ-chain (5Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem... 1992; 267: 16637-16643Google Scholar, 6Farrell D.H. Thiagarajan P. J. Biol. Chem... 1994; 269: 226-231Google Scholar), peptides containing an RGD motif are competitive inhibitors of fibrinogen binding to αIIbβ3 (4Gartner T.K. Bennett J.S. J. Biol. Chem... 1985; 260: 11891-11894Google Scholar). Moreover, despite chemical cross-linking experiments suggesting that the γ-chain and RGD-containing peptides bind to different subunits of the αIIbβ3 heterodimer (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar), competitive binding measurements indicate that the peptides bind to αIIbβ3 in a mutually exclusive manner (39Bennett J.S. Shattil S.J. Power J.W. Gartner T.K. J. Biol. Chem... 1988; 263: 12948-12953Google Scholar), implying either that the peptides bind to same site or that the binding sites interact allosterically. Hu et al. (40Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem... 1999; 274: 4633-4639Google Scholar), using plasmon resonance spectroscopy to study the effect of RGD ligands on fibrinogen binding to αIIbβ3, concluded that fibrinogen and RGD ligands bind to separate sites on αIIbβ3, but suggested that there is an allosteric relationship between the two. Using chimeras of RGD-insensitive rat αIIbβ3 and RGD-sensitive human αIIbβ3, we found that sensitivity to the inhibitory effects of the tetrapeptide RGDS was determined by the origin of the third and fourth amino-terminal repeats of αIIb. We also found little difference in the affinity of αIIbβ3 on human and rat platelets for fibrinogen. Thus, our data suggest that rather than directly affecting fibrinogen binding, species differences in the third and fourth αIIb repeats affect an allosteric change that regulates fibrinogen binding to αIIbβ3. Ligand binding to αIIbβ3 is thought to involve regions located in the amino-terminal portions of both αIIb and β3 (8Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem... 2000; 275: 7795-7802Google Scholar), although much of this evidence is indirect. The β3 region encompasses amino acids 95–223 (9Lin E.C.K. Ratnikov B.I. Tsai P.M. Carron C.P. Myers D.M. Barbas III, C.F. Smith J.W. J. Biol. Chem... 1997; 272: 23912-23920Google Scholar) and includes the RGD-cross-linking site located in the vicinity of amino acids 109–171 (38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar) as well as an array of oxygenated residues whose fold may resemble that of the ligand-bindingmetal ion-dependentadhesion sites (MIDAS) present in integrin I domains (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar). It is noteworthy that the deleterious effect of an Arg214 → Trp mutation, located in the midst of this sequence, can be reversed by exposing αIIbβ3 to DTT, suggesting that the presence of Trp at residue 214 does not prevent fibrinogen binding to αIIbβ3 directly, but rather obscures the fibrinogen-binding site (41Kouns W. Steiner B. Kunicki T. Moog S. Jutzi J. Jennings L. Cazenave J. Lanza F. Blood.. 1994; 84: 1015-1108Google Scholar). It is also noteworthy that the location of the binding site for RGD-containing peptides in integrins is uncertain, and there is evidence for binding sites in both α- and β-subunits. For example, proteins corresponding to the fourth through seventh amino-terminal repeats of α5 and αIIb bind to fibronectin III fragment-(8–10) and to fibrinogen, respectively, in an RGD- dependent manner (42Baneres J.L. Roquet F. Green M. LeCalvez H. Parello J. J. Biol. Chem... 1998; 273: 24744-24753Google Scholar, 43Gulino D. Boudignon C. Zhang L. Concord E. Rabiet M.-J. Marguerie G. J. Biol. Chem... 1992; 267: 1001-1007Google Scholar). Conversely, experiments using chemical and photoaffinity cross-linking, site-directed mutagenesis, synthetic integrin and RGD-containing peptides, and mAbs have identified regions in the amino-terminal portion of β1- and β3-subunits that recognize the RGD motif (11Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem... 1996; 271: 21978-21984Google Scholar, 38D'Souza S.E. Ginsberg M.H. Lam S.C. Plow E.F. J. Biol. Chem... 1988; 263: 3943-3951Google Scholar,44Bitan G. Scheibler L. Greenberg Z. Rosenblatt M. Chorev M. Biochemistry.. 1999; 38: 3414-3420Google Scholar, 45Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem... 1997; 272: 17283-17292Google Scholar, 46Humphries J.D. Askari J.A. Zhang X.P. Takada Y. Humphries M.J. Mould A.P. J. Biol. Chem... 2000; 275: 20337-20345Google Scholar). Based on these observations, one possible explanation for our results is simply that RGDS does not bind to either rat αIIb or rat β3. However, we found that first, the sensitivity of αIIbβ3 composed of human subunits or of a human α-subunit and rat β-subunit to RGDS was equivalent, and second, binding of mAb 10-758 to human β3 was induced by RGDS to an equal extent regardless of whether αIIb was human, rat, or a human-rat chimera. Thus, our data imply that RGDS binds to both human and rat αIIbβ3 and that differences in its inhibitory potency are due to differences in allosteric events that follow RGDS binding. The portion of αIIb implicated in ligand binding has also been localized to the amino-terminal third of the molecule (15Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem... 1996; 271: 2033-2039Google Scholar) and includes the fibrinogen γ-chain peptide-cross-linking site at amino acids 294–314 (16D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem... 1990; 265: 3440-3446Google Scholar). In addition, a number of naturally occurring and laboratory-induced mutations involving amino acids 145, 183, 184, 189, 190, 191, 193, and 224 have been described that impair αIIbβ3 function, suggesting that these residues interact with αIIbβ3 ligands (18Kamata T. Irie A. Tokuhira M. Takada Y. J. Biol. Chem... 1996; 271: 18610-18615Google Scholar, 19Grimaldi C.M. Chen F. Wu C. Weiss H.J. Coller B.S. French D.L. Blood.. 1998; 91: 1562-1571Google Scholar, 20Tozer E.C. Baker E.K. Ginsberg M.H. Loftus J.C. Blood.. 1999; 93: 918-924Google Scholar). Of note, residues 183–224 are located in the third αIIb repeat (25Poncz M. Eisman R. Heidenreich R. Silver S.M. Vilaire G. Surrey S. Schwartz E. Bennett J.S. J. Biol. Chem... 1987; 262: 8476-8482Google Scholar). Because our data suggest that the third repeat is involved in the allosteric regulation of fibrinogen binding to αIIbβ3, it is possible that mutation of the residues listed above interferes with this allosteric change, rather than directly perturbing the fibrinogen-binding site. The tertiary structure of integrins has yet to be determined. Based on computer modeling, Springer (31Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 65-72Google Scholar) proposed that the amino-terminal portion of integrin α-subunits folds into a seven-bladed β-propeller configuration, with each of the blades corresponding to a β-sheet formed from four anti-parallel β-strands located within each of the amino-terminal repeats. Loops connecting the β-strands would be located on either the upper or low surface of the proposed propeller such that residues in three loops in human αIIbbetween Arg147 and Tyr166, Val182and Leu195, and His215 and Gly233, connecting portions of the third and fourth propeller blades, would be juxtaposed in one quadrant of the upper surface of the propeller (21Basani R.B. French D.L. Vilaire G. Brown D.L. Chen F. Coller B.S. Derrick J.M. Gartner T.K. Bennett J.S. Poncz M. Blood.. 2000; 95: 180-188Google Scholar). Comparison of the amino acid sequence of the loops in human αIIb with that of the analogous portions of rat αIIb (47Thornton M.A. Poncz M. Blood.. 1999; 94: 3947-3950Google Scholar) indicates that the putative second loop is fully conserved, whereas the first and third loops would be only 50% homologous. Thus, it is possible that amino acid sequence differences between human and rat αIIb in the putative first and third loops could be responsible for the differences in sensitivity of human and rat αIIbβ3 to RGD-containing peptides. Alterations in the tertiary and/or quaternary structure of integrins regulate their affinity, and possibly their avidity, for ligands. Recent nuclear magnetic resonance spectroscopic and x-ray crystallographic studies of the I domain of αL emphasize the importance of changes in the conformation of the α-subunit amino terminus in integrin function (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar, 49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). I domains are present in nine integrin α-subunits, where they are inserted between the second and third amino-terminal repeats (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar). In αL and αM, ligands such as ICAM-1–3 (intercellular adhesionmolecule) bind to a divalent cation-containing MIDAS motif on the upper I domain surface (50Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure.. 1995; 3: 1333-1340Google Scholar, 51Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 10277-10281Google Scholar, 52Liddington R. Bankston L. Structure.. 1998; 6: 937-938Google Scholar, 53Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 2215-2220Google Scholar). In the I domain of αL, residues distal to the MIDAS motif, lining a cleft formed by the seventh α-helix and the central β-sheet, regulate ligand binding to αLβ2 allosterically (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar) and constitute the binding site for the αLβ2 inhibitor lovastatin (48Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol... 1999; 292: 1-9Google Scholar). In addition, mutations in the amino- and carboxyl-terminal linker sequences that connect the I domain to the rest of αLeither activate or inactivate I domain function (49Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 5231-5236Google Scholar), implying that the changes in I domain conformation that regulate its function are transmitted from the amino-terminal portion of αL to the I domain via these sequences. In the case of αIIbβ3, agonist-induced changes in tertiary structure are essential for its function (2Bennett J.S. Trends Cardiovasc. Med... 1996; 16: 31-36Google Scholar). Our results indicate that an allosteric change in the third and fourth amino-terminal repeats of αIIb, a portion of αIIb located immediately downstream from the I domain insertion site in I domain-containing integrins, regulates ligand binding to αIIbβ3. Thus, by extrapolation, our data suggest that allosteric changes involving the third and fourth α-subunit repeats may be a general mechanism by which ligand binding to integrins is regulated. We thank Dr. H. Miyazaki for providing mAb P34 and Dr. Bohumil Bednar (Merck Research Laboratories) for providing mAb 10-758. Chinese hamster ovary monoclonal antibody fluorescein isothiocyanate dithiothreitol
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