Effects of Ligand-Mimetic Peptides Arg-Gly-Asp-X (X = Phe, Trp, Ser) on αIIbβ3 Integrin Conformation and Oligomerization

Hantgan, Roy R.; Paumi, Christian M.; Rocco, Mattia; Weisel, John W.

doi:10.1021/bi9907680

Cited by 100 publications

(170 citation statements)

References 71 publications

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“…Platelet stimulation by agonists increases the number of ␣IIb␤3 molecules with accessible fibrinogen-binding sites, but has no effect on the bond strength between fibrinogen and the activated form of the integrin. Thus, our results demonstrate that ␣IIb␤3 activation is an all-or-none phenomenon; each ␣IIb␤3 molecule resides on the platelet in either a completely on or a completely off conformation, which is consistent with structural data (32)(33)(34).…”

Section: Discussionsupporting

confidence: 89%

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Litvinov

Shuman

Bennett

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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172

188

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Integrin activation states determine the ability of these receptors to mediate cell-matrix and cell-cell interactions. The prototypic example of this phenomenon is the platelet integrin, ␣IIb␤3. In unstimulated platelets, ␣IIb␤3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables ␣IIb␤3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified ␣IIb␤3 or ␣IIb␤3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80 -100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single ␣IIb␤3 molecules, indicating that there are only two states of ␣IIb␤3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation͞inactivation state.T he detailed biochemistry of receptor-ligand interactions can be determined from solution and͞or surface studies, but these results do not take into account the response of receptormediated cell functions to externally applied forces encountered in vivo. This consideration is particularly relevant for integrins because of the cellular capacity to regulate the state of integrin activation. The extent to which cells regulate integrin function is highly variable. In some cellular environments, the ability of a specific integrin to support cellular adhesion may be constitutive, whereas in others, the integrin may be inactive or only partially active (1). Thus, it has been concluded that integrins exist in a variety of activation states, although the basis for this conclusion has generally been indirect, based on whole-cell adhesion assays and the interaction of integrins with specific monoclonal antibodies. Accordingly, direct studies of the activation states of individual receptors are important. In addition, such studies can reveal the relative contribution to integrin regulation of changes in receptor conformation (affinity modulation) vs. receptor clustering (avidity; ref. 2).The platelet integrin ␣IIb␤3 (GPIIb͞IIIa), which is inactive on resting platelets but is activated by agonists such as ADP and thrombin, is the prototypic example of adhesion receptor modulation. This tight regulation of ␣IIb␤3 activity is imperative to prevent the spontaneous formation of thrombi. In this paper, we demonstrate a model employing laser tweezers to determine the force between single ligand-receptor bonds eithe...

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Section: Discussionsupporting

confidence: 89%

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Litvinov

Shuman

Bennett

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

172

188

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“…Only the open conformation of the headpiece was seen when ligand was bound, suggesting that in the absence of crystal lattice contacts, ligand binding induced (i) a swing-out of the hybrid domain, and (ii) disruption of the headpiece-tailpiece interface in which the hybrid domain is prominent, leading to integrin extension (5). Lower resolution rotary shadowing EM studies from another group led to a completely different conclusion, that binding of cyclic RGD peptide results in separation of the ␣ and ␤ subunits in the headpiece and that separation between the ␣-subunit ␤-propeller domain and the ␤-subunit I-like domain induces the high-affinity state (6,7). Based on the negative stain EM studies, we hypothesized that the opening of the angle between the I-like domain and the hybrid domain in ␤ subunit is the key feature of the high-affinity conformer.…”

mentioning

confidence: 99%

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Luo

Springer

Takagi

2003

Proc. Natl. Acad. Sci. U.S.A.

145

186

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The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligandbinding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV␤3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the ␤3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both ␤3 and ␤1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.I ntegrins are a family of Ϸ25 cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions and govern migration and anchorage of almost all kinds of cells. Integrins are noncovalently associated ␣␤ subunit heterodimers. Each subunit contains a large N-terminal extracellular domain, a transmembrane segment, and a cytoplasmic C-terminal tail. One unique aspect of integrin function is that the affinity for biological ligand can be up-regulated by a process termed inside-out signaling (1). This is particularly important during activation of leukocytes and platelets, where integrins are converted to high-affinity receptors in Ͻ1 s. It has been suggested that this rapid affinity up-regulation is accomplished by a conformational change in integrin extracellular domains triggered by cytoplasmic signaling pathways after cellular activation.Several alternative models for conformational change have been proposed. Crystal structures obtained of the extracellular domain of ␣V␤3 in Ca 2ϩ (2) or by subsequent soaking in Mn 2ϩ and a cyclic Arg-Gly-Asp (RGD) peptide (3) revealed an unexpected bent conformation, in which the headpiece is folded over and makes extensive contacts with the tailpiece (Fig. 1A). Upon ligand binding, no change was seen at the interface between the I-like and hybrid domains, and only a slight rotation between the ␤-propeller and I-like domains and small movements within the I-like domain were seen. It was hypothesized that both the liganded and unliganded bent structures were in the active state (2, 3). In contrast, NMR, negative stain electron microscopy (EM) with image averaging, ligand binding, physicochemical, and mutational studies show that the bent conformation is physiologic and has low ligand-binding affinity and that activation results in a switchblade-like opening to an extended conformation (4, 5). Two extended conformers were seen that differed...

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“…It is difficult to speculate on the nature of the conformational change induced by these ligands. The GP IIb-IIIa structure has been modeled using electron microscopic and other biochemical data (11,30). The model suggests a very broad interface between the two subunits, any part of which may be involved in the interactions which govern SDS stability.…”

Section: Discussionmentioning

confidence: 99%

“…This is in contrast to dimerization of the glycophorin transmembrane domains, which are also stable to SDS treatment (25). Interestingly, addition of RGDX peptides promote an opening of the structure and less interaction between the two subunit chains (11). However, the conformations induced by RGDX peptides and XP280 appear to be different based on 1) the stability to SDS treatment, 2) the rate of proteolysis in the presence of compound, 3) the stability of the altered conformation in the absence of ligand, and 4) the reversibility of SDS stability by RGDS peptides.…”

Section: Discussionmentioning

confidence: 99%

“…Platelet activation results in a conformation of GP IIb-IIIa that has higher affinity and is competent to bind soluble plasma fibrinogen and other RGDS containing ligands (6). Binding of ligands to GP IIb-IIIa induces conformational changes, which have been studied by proteolysis patterns (7,8), sucrose gradient ultracentrifugation (9), fibrinogen binding measurements (10), light scattering and electron microscopy (11), and antibody recognition of ligand-induced binding sites (LIBS) (12)(13)(14)(15)(16). Here we present evidence that some ligands can induce unusually stable conformations and that these conformations can be long-lived even after removal of the ligand.…”

mentioning

confidence: 99%

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Unusually Stable and Long-lived Ligand-induced Conformations of Integrins

Zolotarjova

Hollis

Wynn

2001

Journal of Biological Chemistry

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Integrins are a large family of cell surface receptors that are involved in a wide range of biological processes. The integrin ␣ IIb ␤ 3 (glycoprotein IIb-IIIa) is a major platelet glycoprotein heterodimeric receptor that mediates platelet aggregation and is currently a target for pharmaceutical intervention. Ligand binding to the receptor has been shown to induce conformational changes by physical methods and the exposure of neoepitopes (the ligand-induced binding sites). Here we show that the antagonist XP280 induces a conformation that is stable to treatment with SDS and that the protein retains this conformation for several days even after dissociation of the inhibitor. These ligand-induced conformational changes take place with purified protein and on intact platelets. They are competable with an RGDS peptide and are stable to reduction but not boiling or treatment with EDTA. The retention of an altered conformation in the absence of the ligand implies the possibility of ligand-induced alteration of biological function even in the absence of ligand. Finally, similar behavior is observed with the integrin ␣ v ␤ 3 , suggesting that access to SDS stable conformations may be conserved throughout the integrin superfamily. The unusual stability, long-lived nature, and potential generality of these conformations could have profound implications for integrin biology.

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Effects of Ligand-Mimetic Peptides Arg-Gly-Asp-X (X = Phe, Trp, Ser) on αIIbβ3 Integrin Conformation and Oligomerization

Cited by 100 publications

References 71 publications

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Unusually Stable and Long-lived Ligand-induced Conformations of Integrins

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