Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Hato, Takaaki; Pampori, Nisar A.; Shattil, Sanford J.

doi:10.1083/jcb.141.7.1685

Cited by 223 publications

(204 citation statements)

References 68 publications

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“…To dissect the role of conformational change from that of receptor proximity, a CID strategy was employed. [195] A gene encoding two copies in tandem of FKBP was fused to that encoding αIIb; the latter is a partner in the integrin complex αIIb-β3. In cells producing the resulting fusion protein, the addition of a dimerizer, AP1510, elicits integrin clustering.…”

Section: 1a Integrins-integrinsmentioning

confidence: 99%

Synthetic Multivalent Ligands as Probes of Signal Transduction

2006

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Cell surface receptors acquire information from the extracellular environment and coordinate intracellular responses. Evidence from biochemical and structural studies indicates that many receptors do not operate as individual entities, but rather as part of higher-order complexes (e.g. dimers and oligomers). Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses. Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades. Despite the proposed importance of receptor-receptor processes in cellular signaling, questions concerning the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication remain unanswered.Chemical synthesis can provide a variety of compounds with which to address the role of receptor assembly in signal transduction. The focus of this review is one such approach -the use of synthetic multivalent ligands to characterize receptor function. Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements. Their unique architectures imbue multivalent ligands with the ability to access binding modes not available to monovalent compounds. Multivalent ligands, therefore, are capable of illuminating aspects of inter-receptor processes that are not readily probed using conventional approaches. We suggest that, as focus shifts from investigations of the function of individual proteins and toward the analysis of multi-receptor signaling complexes, multivalent ligands will become even more valuable tools with which to ask sophisticated mechanistic questions. Further, multivalent ligands may provide new opportunities for manipulating receptor systems for the deconvolution of pathways, diagnosis, and, ultimately, the treatment of disease.

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Section: 1a Integrins-integrinsmentioning

confidence: 99%

Synthetic Multivalent Ligands as Probes of Signal Transduction

2006

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“…As shown in Fig. 6, an overlay of the 15 N-HSQC spectra for 15 N-labeled ␤3 TM-CYTO, and for the mixture of labeled ␤3 TM-CYTO with unlabeled ␣IIb TM-CYTO, revealed no significant changes in chemical shift. Peaks were essentially superimposable: typical chemical shift changes in the proton dimension were less than 0.02 ppm and the largest change was Ϸ0.08 ppm.…”

Section: Ultracentrifugation Of Tm-cyto Proteins In Dpc Micellesmentioning

confidence: 99%

“…NMR Spectroscopy. 15 N-labeled TM-CYTO proteins were mixed with deuterated DPC (Cambridge Isotope Laboratories, Cambridge, MA) before being dissolved in 650 l of freshly made 50 mM deuterated imidazole͞100 mM KCl͞2 mM MgCl 2 , pH 7.40. CaCl 2 (2 mM) was added when needed.…”

Section: Dispersion Of the ␣Iib And ␤3 Tm-cyto Proteins In Dodecylphomentioning

confidence: 99%

“…In B lymphocytes, low concentrations of cytochalasin D increase cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and concurrently increase the rate of random diffusion of the integrin ␣L␤2 in the lymphocyte membrane, suggesting that the formation of ␣L␤2 oligomers either accompanies or is responsible for the function of this integrin (13). Ligand-occupied clusters of ␣IIb␤3 are present on the surface of thrombin-stimulated platelets (14), and the forced clustering of ␣IIb␤3 expressed in Chinese hamster ovary cells has been found to induce ligand binding, albeit to a lesser extent than exposing ␣IIb␤3 to stimulatory mAbs (15). Nevertheless, these data suggest that the formation of ␣IIb␤3 oligomers after agonist stimulation may contribute to the regulation of ␣IIb␤3 function.…”

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confidence: 99%

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Oligomerization of the integrin αIIbβ3: Roles of the transmembrane and cytoplasmic domains

Babu

Lear

et al. 2001

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

162

165

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Integrins are a family of ␣͞␤ heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the ␣ and ␤ subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of ␣IIb␤3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the ␣ and ␤ subunits of ␣IIb␤3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the ␣-and ␤-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.I ntegrins, a family of ␣͞␤ heterodimers, mediate essential cell-cell and cell-matrix interactions (1). Each subunit of the integrin heterodimer is composed of a large extracellular domain, a transmembrane (TM) helix, and a short cytoplasmic (CYTO) tail. Heterodimer formation results from interactions between sequences located in the extracellular domain of each subunit (2). Many cells actively regulate integrin ligand-binding activity (3). The prototypic example of integrin regulation is the platelet integrin ␣IIb␤3 (4). In unstimulated platelets, ␣IIb␤3 is inactive, whereas exposing platelets to agonists such as ADP and thrombin enables ␣IIb␤3 to bind ligands such as fibrinogen and von Willebrand factor. The integrin is activated in a bidirectional manner, in which intracellular events can trigger a conformational change in the extracellular ligand-binding domains (inside-out signaling) or vice versa. In one proposed mechanism for this process, the CYTO tails of ␣IIb and ␤3 interact in the inactive state through the formation of a salt bridge (5). This interaction is broken and the CYTO domains separate when the integrin is activated. Evidence for this hypothesis came from mutational studies (5), as well as biochemical studies that seemed to show a weak but divalent cation-dependent interaction between peptides corresponding to the CYTO tails of ␣IIb and ␤3 (6, 7). Further, elegant protein engineering studies by Springer and coworkers (8,9) unambiguously demonstrated that when the CYTO domains or the C termini of the extracellular domains were forced to interact, the integrin ␣L␤2 or ␣5␤1 was inactivated. However, a very recent and carefully executed NMR study indicated that the ␣IIb and ␤3 CYTO tails were unable to interact, even when tethered in close proximity from the same end of a heterodimeric coiled coil (10). Further, the observation that replaci...

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“…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).…”

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confidence: 99%

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.

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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Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Cited by 223 publications

References 68 publications

Synthetic Multivalent Ligands as Probes of Signal Transduction

Synthetic Multivalent Ligands as Probes of Signal Transduction

Oligomerization of the integrin αIIbβ3: Roles of the transmembrane and cytoplasmic domains

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

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