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• W1817963979 abstract "Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca2+ and Mg2+ bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin αIIbβ3. The library was used to select for peptides that bind to the integrin αIIbβ3 preferentially in Ca2+ versus Mg2+. Peptides were identified which bound selectively in each ion. The Ca2+-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg2+-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg2+-selected peptides contained a unique basic patch that protrudes from the base of the turn. Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca2+ and Mg2+ bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin αIIbβ3. The library was used to select for peptides that bind to the integrin αIIbβ3 preferentially in Ca2+ versus Mg2+. Peptides were identified which bound selectively in each ion. The Ca2+-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg2+-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg2+-selected peptides contained a unique basic patch that protrudes from the base of the turn. ligand-competent inhibitory site fibrinogen high performance liquid chromatography bovine serum albumin enzyme-linked immunosorbent assay dimethyl sulfoxide Integrin-mediated cell adhesion is regulated tightly by extracellular divalent cations such as Ca2+ and Mg2+ (for review, see Ref. 1Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar). Interestingly though, divalent ions can have opposing effects on ligand binding, promoting both cell adhesion and cell detachment (2Staatz W.D. Rajpara S.M. Wayner E.A. Carter W.G. Santoro S.A. J. Cell Biol. 1989; 108: 1917-1924Crossref PubMed Scopus (289) Google Scholar, 3Gailit J. Ruoslahti E. J. Biol. Chem. 1988; 263: 12927-12933Abstract Full Text PDF PubMed Google Scholar, 4Hu D.D. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 9917-9925Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 6Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar). This can be explained by the fact that integrins contain two classes of divalent ion binding sites. One class of sites, called ligand-competent (LC)1 sites, must be filled for ligand to bind (7Smith J.W. Piotrowicz R.S. Mathis D.M. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar). The LC site(s) can bind to a number of different ions including Ca2+, Mg2+, and Mn2+. A number of studies (8D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar), including recent co-crystals of integrin I domains with bound ligands (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar), suggest that the LC sites may actually sit within the ligand binding pocket and make direct contact with ligand.A separate class of ion binding sites is allosteric to the ligand binding site and inhibits ligand binding by increasing the rate of ligand dissociation. Because of their effects on ligand dissociation, we called these allosteric sites inhibitory, or “I,” sites (10Hu D.D. Barbas C.F.I. Smith J.W. J. Biol. Chem. 1996; 271: 21745-21751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The I site(s) appear to bind selectively to Ca2+. The effects of the I site can be pronounced, as an integrin with Ca2+ bound at the I site can have a dissociation rate that is 20-fold higher than when the I site is unoccupied. Because the affinities of the LC and I sites for divalent ions are within the physiologic ranges, these sites act cooperatively, and in an opposing manner, to regulate the function of the integrin ligand binding pocket.There are several physiologic and pathophysiologic circumstances in which divalent ions play an important role in regulating cell adhesion. For example, osteoclasts, the primary bone-resorbing cell, adhere to the bone surface through the αvβ3 integrin. The adherent osteoclast resorbs mineralized bone, liberating free Ca2+, which can rise above 10 mm (11Silver I.A. Murrills R.J. Etherington D.J. Exp. Cell Res. 1988; 175: 266-277Crossref PubMed Scopus (725) Google Scholar). This elevation in [Ca2+] is then sensed by the osteoclast via a receptor (12Miyauchi A. Hruska K.A. Greenfield E.M. Duncan R. Alvarez J. Baratollo R. Colucci S. Zambonin-Zallone A. Teitelbaum S.L. Teti A. J. Cell Biol. 1990; 111: 2543-2552Crossref PubMed Scopus (197) Google Scholar, 13Zaidi M. Kerby J. Huang C.L. Alam T. Rathod H. Chambers T.J. Moonga B.S. J. Cell. Physiol. 1991; 149: 422-427Crossref PubMed Scopus (84) Google Scholar), and this ultimately causes the osteoclast to detach from the bone surface. The identity of this osteoclast Ca2+ receptor remains unclear, but it appears to have many of the properties of the allosteric I site on the αvβ3 integrin (4Hu D.D. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 9917-9925Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Hu D.D. Barbas C.F.I. Smith J.W. J. Biol. Chem. 1996; 271: 21745-21751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Consequently, we proposed that the liberated Ca2+ binds to this site on integrin, accounting for the effects of this ion on osteoclast detachment. A similar mechanism can be proposed in wound healing, where keratinocyte integrins play a major role. In wound fluids the ratio of Mg2+ to Ca2+ is substantially higher than under normal circumstances (14Grzesiak J.J. Pierschbacher M.D. J. Clin. Invest. 1995; 95: 227-233Crossref PubMed Scopus (104) Google Scholar). This scenario is expected to favor the action of the integrin LC ion binding sites over the I site and favor adhesion over detachment. Indeed, the elevated levels of Mg2+ appear to promote integrin-mediated migration of keratinocytes and healing of the wounds (14Grzesiak J.J. Pierschbacher M.D. J. Clin. Invest. 1995; 95: 227-233Crossref PubMed Scopus (104) Google Scholar).Divalent ions also influence the ligand binding properties of the integrin that is the subject of this study, platelet integrin αIIbβ3 (15Kirchhofer D. Gailit J. Ruoslahti E. Grzesiak J. Pierschbacher M.D. J. Biol. Chem. 1990; 265: 18525-18530Abstract Full Text PDF PubMed Google Scholar). The αIIbβ3 integrin is somewhat unique in that it contains two physically distinct but interacting ligand binding pockets (16Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem. 1999; 274: 4633-4639Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). One ligand binding pocket binds to the carboxyl-terminal region of fibrinogen (Fg), called the γ-chain site. The binding of αIIbβ3 to the γ-chain is necessary for the aggregation of platelets in blood (17Kloczewiak M. Timmons S. Hawiger J. Biochem. Biophys. Res. Commun. 1982; 107: 181-187Crossref PubMed Scopus (104) Google Scholar, 18Kloczewiak M. Timmons S. Hawiger J. Thromb. Res. 1983; 29: 249-255Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 19Farrell D.H. Thiagarajan P. Chung D.W. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10729-10732Crossref PubMed Scopus (297) Google Scholar). Importantly, the γ-chain of Fg lacks the RGD motif. Interestingly, the effects of divalent ions on the binding of the γ-chain to αIIbβ3 are different from the effects on RGD binding. Binding between the Fg γ-chain and αIIbβ3 proceed faster in Ca2+than in any other ion, indicating that the I site is not involved in this interaction.Integrin αIIbβ3 also contains a separate RGD binding pocket that binds to ligands such as von Willebrand factor, fibronectin, as well as RGD peptides and their mimetics. This RGD binding site is governed cooperatively by the LC and I ion binding sites as discussed above. Another important feature of the RGD binding site is that it is allosterically connected to the Fg γ-chain binding site. When αIIbβ3 is occupied with RGD ligands, the rate of dissociation for Fg is increased dramatically. Altogether then, the ligand binding properties of αIIbβ3 are regulated in a complex manner, with both divalent ions and the presence of additional ligands making key contributions. In general though, the current understanding of these interactions suggests that platelet aggregation to soluble Fg will be enhanced by Ca2+ and that platelet adhesion to immobilized RGD ligands such as von Willebrand factor will be enhanced by Mg2+.Given the clinical importance of dissecting these two functions of αIIbβ3 and the lack of a structural basis for ion-selective binding to integrins, we sought to engineer RGD ligands for αIIbβ3 which exhibit Mg2+- and Ca2+-selective binding. To address these issues we constructed a phage library of cyclic RGD peptides of unparalleled complexity. The library is comprised of ninemers that are conformationally constrained by two terminal cysteine residues. The RGD motif and six randomized residues lie between the two cysteines. The library takes full access of all possible conformations of RGD by displaying the motif in every register. In solution, all of the peptides that bind to αIIbβ3 adopt the conformation of a general β-turn, with the RGD sequence near the apex of the turn. Yet, the study reveals key differences in the primary structure of ion-selective RGD peptides. A distinguishing feature of the Mg2+-selective peptides is the presence of a protruding basic patch adjacent to the turn.DISCUSSIONThe RGD motif is the key recognition element for a large segment of the integrin protein family. However, there is an underlying binding specificity between integrins and RGD for which we still have no structural basis. In most cases, integrin binding RGD motifs are presented at the apex of β-turns (36Krezel A.M. Ulmer J.S. Wagner G. Lazarus R.A. Protein Sci. 2000; 9: 1428-1438Crossref PubMed Scopus (20) Google Scholar, 37Kodandapani R. Veerapandian B. Kunicki T.J. Ely K.R. J. Biol. Chem. 1995; 270: 2268-2273Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 38Leahy D.J. Erickson H.P. Aukhil I. Joshi P. Hendrickson W.A. Proteins. 1994; 19: 48-54Crossref PubMed Scopus (74) Google Scholar, 39Dickinson C.D. Veerapandian B. Dai X.P. Hamlin R.C. Xuong N.H. Ruoslahti E. Ely K.R. J. Mol. Biol. 1994; 236: 1079-1092Crossref PubMed Scopus (183) Google Scholar, 40Sutcliffe M.J. Jaseja M. Hyde E.I. Lu X. Williams J.A. Nat. Struct. Biol. 1994; 1: 802-807Crossref PubMed Scopus (46) Google Scholar, 41Assa-Munt N. Jia X. Laakkonen P. Ruoslahti E. Biochemistry. 2001; 40: 2373-2378Crossref PubMed Scopus (134) Google Scholar). Yet, this body of information falls short of providing a systematic data set of key structure-activity relationships because the RGD loops are embedded within different protein backbones. We reasoned that such data could be obtained by using biochemical selection to obtain RGD loops with a given biochemical property and then probing the solution structures of these loops with NMR. As a test of the strategy, we created the most diverse library of RGD loops assembled to date and used this library to identify RGD loops that distinguish subtle conformational differences in the ligand binding pocket of a single integrin. Our findings indicate that even subtle differences in the shape of a single RGD binding pocket can be discerned using a library of sufficient diversity.We screened for RGD motifs that can distinguish the αIIbβ3 integrin when its cation binding sites are occupied with either Ca2+ or Mg2+. RGD loops were identified with both biochemical properties. Loops from both selections could be grouped into well defined subfamilies with rather obvious consensus motifs. There are several common features among the peptides selected in the two ions. First, the RGD motif is observed most frequently at positions 4–6 and 5–7, slots that put the RGD at the apex of a loop or β-turn. Second, most of the RGD motifs contain at least one, and often two, aromatic residues. In the vast majority of cases an aromatic residue follows directly after RGD. Tryptophan, phenylalanine, and tyrosine residues were all observed at this position. This observation is consistent with prior reports showing that the affinity of linear RGD peptides for αIIbβ3 is enhanced greatly by inclusion of a hydrophobic residue just after RGD (42Plow E.F. Pierschbacher M.D. Ruoslahti E. Marguerie G.A. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8057-8061Crossref PubMed Scopus (441) Google Scholar, 43Ruggeri Z.M. Houghten R.A. Russell S.R. Zimmerman T.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5708-5712Crossref PubMed Scopus (93) Google Scholar). Aromatic residues were often found at other positions, both preceding and following the RGD motif.Despite the similarity in the positioning of RGD within the loops, there are other features that distinguish the two functional families. The primary distinguishing feature of the Mg2+-selected loops is the presence of two basic, or polar, residues that fall on the carboxyl-terminal side of the RGD. Two subfamilies of phage from the Mg2+ selection display this feature (Table III). Both subfamilies contain the RGD at P4–6. The small distinctions arise at P7, which is occupied by a hydrophobic residue in one family and an aromatic residue in the other. There is also a distinction at P8, which is populated exclusively by Arg or Lys in the first subfamily but can be occupied by noncharged, but usually polar, residues in the second family. The NMR structures of peptides that represent both subfamilies, D16 and D18, show the basic patch to protrude from the base of the RGD loop. The findings presented here suggest that this patch is necessary for Mg2+-selective binding.The inherent flexibility of the loops is a second feature that appears to distinguish the Ca2+- and Mg2+-selective peptides. The two phage loops with the highest binding affinity and the highest degree of selectivity for binding in Ca2+ are A13 and A22. Both of these loops contain two additional cysteine residues that presumably constrain the peptide further by forming an additional disulfide bond. This extra disulfide is expected to impose more rigidity onto the RGD loop, and the NMR data for A22 support this concept.The NMR spectra for the peptides containing a single disulfide bridge are also consistent with enhanced flexibility for the Mg2+-selected loops. Both D16 and D18 display fewer NOEs than their Ca2+-selective counterpart, A1. The dearth of NOEs in D16 and D18 may be interpreted as increased conformational flexibility as has been observed for highly flexible or unfolded regions (44Neuhaus D. Williamson M.P. The Nuclear Overhauser Effect in Structural and Conformational Analysis.2nd Ed. Wiley-VCH, New York2000Google Scholar). It is also conceivable that NOEs for D16 and D18 are undetectable under the experimental conditions chosen. We view this latter possibility as unlikely because D16 and D18 are the same size as A1, where NOEs are clearly evident, and all three populate the same NOE regime. Furthermore, studies on D16 and D18 were performed under solvent and temperature conditions identical to those for A1. The strong sequential αN NOEs and large spin-spin coupling constants (3 J HNα above 9 Hz) of the A1 peptide (A1 spectrum and calculations in the supplementary material) may also be interpreted to indicate that this peptide has a more extended conformation (22Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Spin-spin coupling constants were significantly lower for D16 and D18, suggesting enhanced flexibility compared with the Ca2+-selective A1. Although the measurement of reduced heteronuclear NOEs is the method of choice for probing flexibility (45Buck M. Schwalbe H. Dobson C.M. J. Mol. Biol. 1996; 257: 669-683Crossref PubMed Scopus (92) Google Scholar, 46Eliezer D. Yao J. Dyson H.J. Wright P.E. Nat. Struct. Biol. 1998; 5: 148-155Crossref PubMed Scopus (386) Google Scholar) it requires 15N-labeled peptides, which are not available in our case.The presumed flexibility of D16 and D18, superimposed to a local signature provided by their additional cationic patch, may well be an important regulator of the binding of physiologic ligands. Such inherent flexibility would allow for protein-protein interactions subsequent to initial contact with the RGD sequence. The precedent for differential rigidity in RGD-containing loops from homologous cell adhesion domains has been demonstrated for homologous fibronectin type III domains from tenascin and fibronectin (47Carr P.A. Erickson H.P. Palmer A.G. Structure. 1997; 5: 949-959Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the context of small peptidic inhibitors, other examples have been reported in which increased flexibility is needed for binding, e.g. for factor Xa inhibitors (48Duggan B.M. Dyson H.J. Wright P.E. Eur. J. Biochem. 1999; 265: 539-548Crossref PubMed Scopus (46) Google Scholar).Our study also raises another important issue concerning recognition of ligands by the two distinct ligand binding pockets on αIIbβ3. We showed previously that αIIbβ3 contains two distinct ligand binding pockets that are linked allosterically (16Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem. 1999; 274: 4633-4639Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). One binding pocket binds to the physiologic ligand Fg, and the other binding pocket binds to RGD ligands. An important and unappreciated aspect of the allosteric connection between these two binding sites is the fact that RGD ligands can bind to αIIbβ3 even when Fg occupies its binding site. In doing so RGD ligands induce the dissociation of bound Fg. One observation from the present study may relate to this mechanism. Most of the RGD peptides from the current study inhibit Fg with far lower IC50 values than they exhibit for Fab-9. This is expected given that the RGD peptides and Fab-9 are anticipated to bind the same binding pocket and compete for association with integrin. Interestingly however, peptide A1 behaved differently. It inhibited Fab-9 binding at far lower concentrations than required for inhibition of Fg binding. The reason for this difference in behavior is not entirely clear, but one is tempted to speculate that this peptide could actually be a mimic of the Fg γ-chain motif rather than a true RGD mimic. It would then compete directly with Fg for association with αIIbβ3 but not Fab-9. Given the molecular diversity within the RGD superlibrary, nearly 500 million different motifs, it is not unreasonable to expect that some of these motifs could approximate the shape of the fibrinogen γ-chain, a motif of between 11 and 15 residues (18Kloczewiak M. Timmons S. Hawiger J. Thromb. Res. 1983; 29: 249-255Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 49Kloczewiak M. Timmons S. Lukas T.J. Hawiger J. Biochemistry. 1984; 23: 1767-1774Crossref PubMed Scopus (350) Google Scholar). This idea is also consistent with the fact that both peptide A1 and the γ-chain peptide adopt type II β-turns in solution, with an aspartic acid near the apex of the turn.Because αIIbβ3 is an excellent paradigm for most other members of the integrin family, and because many other integrins also exhibit Ca2+- and Mg2+-regulated binding of physiologic ligands, the results presented here could potentially be extrapolated to other integrins. The availability of peptides that bind to the two conformations of αIIbβ3 is likely to make it possible to determine the structures of ion-selective ligands while bound to integrin. A comparison of the peptide solution structures with the integrin-bound structures should provide a structural basis for the way that ions influence ligand binding specificity. Such information will provide additional framework for understanding ion-regulated binding of all integrins.This study also lays the groundwork for two future lines of investigation. First, the ion-selective ligands identified here can be applied to test hypotheses regarding the role of ions in regulating platelet adhesion functions. It is well known that certain RGD-based antagonists have different effects on platelet aggregationversus bleeding times. These differences have often been attributed to a distinction in binding kinetics which is still not understood. We have speculated that thrombosis, which is driven by aggregation, is a Ca2+-controlled event, and that hemostasis, which is driven by adhesion to RGD-containing proteins, is a Mg2+-controlled event. The availability of peptides with well defined selectivity for binding in the two ions should make it possible to test this hypothesis.Finally, the study validates the use of the RGD superlibrary for the selection of high affinity binders with exquisite specificity. We have shown previously that RGD-containing loops can be grafted into proteins, endowing them with integrin binding function. The availability of the superlibrary for identifying highly selective RGD loops should enhance our ability to design proteins that are targeted to specific integrin targets. This type of highly specific target could potentially enhance the therapeutic efficacy of protein-based drugs. Integrin-mediated cell adhesion is regulated tightly by extracellular divalent cations such as Ca2+ and Mg2+ (for review, see Ref. 1Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar). Interestingly though, divalent ions can have opposing effects on ligand binding, promoting both cell adhesion and cell detachment (2Staatz W.D. Rajpara S.M. Wayner E.A. Carter W.G. Santoro S.A. J. Cell Biol. 1989; 108: 1917-1924Crossref PubMed Scopus (289) Google Scholar, 3Gailit J. Ruoslahti E. J. Biol. Chem. 1988; 263: 12927-12933Abstract Full Text PDF PubMed Google Scholar, 4Hu D.D. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 9917-9925Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 6Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar). This can be explained by the fact that integrins contain two classes of divalent ion binding sites. One class of sites, called ligand-competent (LC)1 sites, must be filled for ligand to bind (7Smith J.W. Piotrowicz R.S. Mathis D.M. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar). The LC site(s) can bind to a number of different ions including Ca2+, Mg2+, and Mn2+. A number of studies (8D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar), including recent co-crystals of integrin I domains with bound ligands (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar), suggest that the LC sites may actually sit within the ligand binding pocket and make direct contact with ligand. A separate class of ion binding sites is allosteric to the ligand binding site and inhibits ligand binding by increasing the rate of ligand dissociation. Because of their effects on ligand dissociation, we called these allosteric sites inhibitory, or “I,” sites (10Hu D.D. Barbas C.F.I. Smith J.W. J. Biol. Chem. 1996; 271: 21745-21751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The I site(s) appear to bind selectively to Ca2+. The effects of the I site can be pronounced, as an integrin with Ca2+ bound at the I site can have a dissociation rate that is 20-fold higher than when the I site is unoccupied. Because the affinities of the LC and I sites for divalent ions are within the physiologic ranges, these sites act cooperatively, and in an opposing manner, to regulate the function of the integrin ligand binding pocket. There are several physiologic and pathophysiologic circumstances in which divalent ions play an important role in regulating cell adhesion. For example, osteoclasts, the primary bone-resorbing cell, adhere to the bone surface through the αvβ3 integrin. The adherent osteoclast resorbs mineralized bone, liberating free Ca2+, which can rise above 10 mm (11Silver I.A. Murrills R.J. Etherington D.J. Exp. Cell Res. 1988; 175: 266-277Crossref PubMed Scopus (725) Google Scholar). This elevation in [Ca2+] is then sensed by the osteoclast via a receptor (12Miyauchi A. Hruska K.A. Greenfield E.M. Duncan R. Alvarez J. Baratollo R. Colucci S. Zambonin-Zallone A. Teitelbaum S.L. Teti A. J. Cell Biol. 1990; 111: 2543-2552Crossref PubMed Scopus (197) Google Scholar, 13Zaidi M. Kerby J. Huang C.L. Alam T. Rathod H. Chambers T.J. Moonga B.S. J. Cell. Physiol. 1991; 149: 422-427Crossref PubMed Scopus (84) Google Scholar), and this ultimately causes the osteoclast to detach from the bone surface. The identity of this osteoclast Ca2+ receptor remains unclear, but it appears to have many of the properties of the allosteric I site on the αvβ3 integrin (4Hu D.D. Hoyer J.R. Smith J.W. J. Biol. Chem. 1995; 270: 9917-9925Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Hu D.D. Barbas C.F.I. Smith J.W. J. Biol. Chem. 1996; 271: 21745-21751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Consequently, we proposed that the liberated Ca2+ binds to this site on integrin, accounting for the effects of this ion on osteoclast detachment. A similar mechanism can be proposed in wound healing, where keratinocyte integrins play a major role. In wound fluids the ratio of Mg2+ to Ca2+ is substantially higher than under normal circumstances (14Grzesiak J.J. Pierschbacher M.D. J. Clin. Invest. 1995; 95: 227-233Crossref PubMed Scopus (104) Google Scholar). This scenario is expected to favor the action of the integrin LC ion binding sites over the I site and favor adhesion over detachment. Indeed, the elevated levels of Mg2+ appear to promote integrin-mediated migration of keratinocytes and healing of the wounds (14Grzesiak J.J. Pierschbacher M.D. J. Clin. Invest. 1995; 95: 227-233Crossref PubMed Scopus (104) Google Scholar). Divalent ions also influence the ligand binding properties of the integrin that is the subject of this study, platelet integrin αIIbβ3 (15Kirchhofer D. Gailit J. Ruoslahti E. Grzesiak J. Pierschbacher M.D. J. Biol. Chem. 1990; 265: 18525-18530Abstract Full Text PDF PubMed Google Scholar). The αIIbβ3 integrin is somewhat unique in that it contains two physically distinct but interacting ligand binding pockets (16Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem. 1999; 274: 4633-4639Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). One ligand binding pocket binds to the carboxyl-terminal region of fibrinogen (Fg), called the γ-chain site. The binding of αIIbβ3 to the γ-chain is necessary for the aggregation of platelets in blood (17Kloczewiak M. Timmons S. Hawiger J. Biochem. Biophys. Res. Commun. 1982; 107: 181-187Crossref PubMed Scopus (104) Google Scholar, 18Kloczewiak M. Timmons S. Hawiger J. Thromb. Res. 1983; 29: 249-255Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 19Farrell D.H. Thiagarajan P. Chung D.W. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10729-10732Crossref PubMed Scopus (297) Google Scholar). Importantly, the γ-ch" @default.
• W1817963979 created "2016-06-24" @default.
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• W1817963979 date "2002-03-01" @default.
• W1817963979 modified "2023-09-27" @default.
• W1817963979 title "Selection and Structure of Ion-selective Ligands for Platelet Integrin αIIbβ3" @default.
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