Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3.

Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca(2+) and Mg(2+) 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 alpha(IIb)beta(3). The library was used to select for peptides that bind to the integrin alpha(IIb)beta(3) preferentially in Ca(2+) versus Mg(2+). Peptides were identified which bound selectively in each ion. The Ca(2+)-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 Mg(2+)-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 Mg(2+)-selected peptides contained a unique basic patch that protrudes from the base of the turn.     

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.     

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.     

Probing for integrin alpha v beta3 binding of RGD peptides using fluorescence polarization

Integrin alpha(v)beta(3) is an adhesion molecule involved in tumor invasion, angiogenesis, and metastasis. There is substantial interest in developing novel agents that bind to integrin alpha(v)beta(3). Here we report the synthesis and characterization of a fluorescent integrin alpha(v)beta(3) probe and its use in a nonradioactive, simple, sensitive fluorescence polarization (FP) assay to quantify binding to integrin alpha(v)beta(3). For assay validation, the FP assay was compared to a cell adhesion assay. In the two assays, probe binding to integrin alpha(v)beta(3) showed a similar dependence on probe concentration. The FP assay was successfully applied to measure the binding affinity to integrin alpha(v)beta(3) of several cyclic peptides containing the Arg-Gly-Asp (RGD) motif. The FP assay we describe here may be appropriate for high-throughput screening for integrin alpha(v)beta(3)-binding ligands used for anti-integrin therapy or noninvasive imaging of integrin expression.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.     

Structural analysis of fertilin(beta) cyclic peptide mimics that are ligands for alpha6beta1 integrin.

The NMR structural analysis of two fertilin(beta) mimics cyclo(EC2DC1)YNH2, 1, and cyclo(D2EC2D1C1)YNH2, 2 is described. Both of these mimics are moderate inhibitors of sperm-egg binding with IC50 values of 500 microm in a mouse in vitro fertilization assay. For peptide 1, the optimized conformations that best match the NMR data have a pseudo-type II' beta-turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC2D1C1 sequence is in a nonclassical (type IV) beta-turn. For peptide 2, the conformation that best matches the NMR data has two turns: a pseudo-type II' beta-turn in the D2EC2D1 sequence followed by a nonclassical beta-turn in the EC2D1C1 sequence. The Cbeta-Cbeta distance between E and D1 in peptide 1 is 9.1 A, in peptide 2, it is 7.7 A. Thus, one possibility for the high IC50 values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the alpha6beta1 integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm-egg binding.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling

Integrin alpha(IIb)beta(3) activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin alpha(IIb)beta(3) signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin alpha(IIb)beta(3) in resting platelets and in human embryonal kidney 293 cells expressing alpha(IIb)beta(3). The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin alpha(IIb) is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to alpha(IIb)beta(3) during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac(alpha) in 293 cells decreased alpha(IIb)beta(3)-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac(alpha) expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated alpha(IIb)beta(3) adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac (alpha)-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A(calpha) expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac (alpha) can negatively regulate integrin alpha(IIb)beta(3) signaling by suppressing the ERK1/2 signaling pathway.     

The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.

Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.     

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.     

Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.     

RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin alpha(IIb)beta3

We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplasmic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit α_IIb. These included a hypothetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and confirm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events.     

NMR analysis of structure and dynamics of the cytosolic tails of integrin alpha IIb beta 3 in aqueous solution.

The structural and dynamic properties of the cytosolic tails of the adhesion receptor integrin alphaIIbbeta3, fused to a coiled-coil construct via (Gly)(3) linkers, were studied in aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. Both tails were largely flexible and unstructured, although, in the beta3 tail, residues Arg(724)-Ala(735) have a propensity to form a helical structure and residues Asn(744)-Tyr(747) (NPLY) have a propensity to adopt reverse-turn conformations. The mutation beta3(Y747A) disrupted this reverse-turn tendency and markedly reduced the affinity of the head domain of the cytoskeletal protein, talin for the beta3 tail. Omission of the (Gly)(3) linker connecting the coiled-coiled helices and the integrin tails lead to helix propagation into the beta3 tail extending up to eight residues. A variety of different tail constructs were made and studied to reveal tail-tail interactions, but surprisingly no significant interactions between both tails could be detected within the context of our constructs. These results provide structural insight into a highly conserved beta tail motif (NPXY/F) required for integrin signaling and highlight a second transiently structured region (residues Arg(724)-Ala(735)), which might also be of functional significance.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function

Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.     

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.     

A membrane-distal segment of the integrin alpha IIb cytoplasmic domain regulates integrin activation

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha(IIb)beta(3) cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha(IIb) and beta(3) cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha(IIb)beta(3), indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta(3)(Y747A) mutation into the beta(3) tail. A membrane-distal heptapeptide sequence in the alpha(IIb) tail ((997)RPPLEED) is responsible for this effect on beta(3). Membrane-permeant palmitoylated peptides, containing this alpha(IIb) sequence, specifically blocked alpha(IIb)beta(3) activation in platelets. Thus, this region of the alpha(IIb) tail causes the beta(3) tail to resemble that of beta(3)(Y747A) and suppresses activation of the integrin.