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Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin α IIb β 3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin α IIb β 3 receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

Disabled-2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell growth, trafficking, differentiation, and homeostasis. Emerging evidences place Dab2 as a novel modulator of cell-cell interaction; however, its mode of action has remained largely elusive. In this review, we highlight the relevance of Dab2 function in cell signaling and development and provide the most recent and comprehensive analysis of Dab2's action as a mediator of homotypical and heterotypical interactions. Accordingly, Dab-2 controls the extent of platelet aggregation through various motifs within its N-terminus. Dab2 interacts with the cytosolic tail of the integrin receptor blocking inside-out signaling, whereas extracellular Dab2 competes with fibrinogen for integrin αIIb β3 receptor binding and, thus, modulates outside-in signaling. An additional level of regulation results from Dab2's association with cell surface lipids, an event that defines the extent of cell-cell interactions. As a multifaceted regulator, Dab2 acts as a mediator of endocytosis through its association with the [FY]xNPx[YF] motifs of internalized cell surface receptors, phosphoinositides, and clathrin. Other emerging roles of Dab2 include its participation in developmental mechanisms required for tissue formation and in modulation of immune responses. This review highlights the various novel mechanisms by which Dab2 mediates an array of signaling events with vast physiological consequences., (© 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.)

The Wnt-dependent, β-catenin-independent pathway modulates cell movement and behavior. A downstream regulator of this signaling pathway is Dishevelled (Dvl), which, among other multiple interactions, binds to the Frizzled receptor and the plasma membrane via phosphatidic acid (PA) in a mechanism proposed to be pH-dependent. While the Dvl DEP domain is central to the β-catenin-independent Wnt signaling function, the mechanism underlying its physical interaction with the membrane remains elusive. In this report, we elucidate the structural and functional basis of PA association to the Dvl2 DEP domain. Nuclear magnetic resonance, molecular-dynamics simulations, and mutagenesis data indicated that the domain interacted with the phospholipid through the basic helix 3 and a contiguous loop with moderate affinity. The association suggested that PA binding promoted local conformational changes in helix 2 and β-strand 4, both of which are compromised to maintain a stable hydrophobic core in the DEP domain. We also show that the Dvl2 DEP domain bound PA in a pH-dependent manner in a mechanism that resembles deprotonation of PA. Collectively, our results structurally define the PA-binding properties of the Dvl2 DEP domain, which can be exploited for the investigation of binding mechanisms of other DEP domain-interacting proteins., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at -80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α(IIb)β(3) integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.

Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from endocytosis to platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the inner leaflet of the plasma membrane. As a result of platelet activation, Dab2 is released from α-granules and associates with both the αIIbβ3 integrin receptor and sulfatide lipids on the platelet surface through its N-terminal region including the PTB domain (N-PTB), thus, modulating platelet aggregation. Thrombin, a strong platelet agonist, prevents Dab2 function by cleaving N-PTB within the two basic motifs required for sulfatide association, a reaction that is prevented when Dab2 is bound to these sphingolipids. We have characterized the membrane binding properties of Dab2 N-PTB using micelles enriched with Dab2 lipid ligands, sulfatides and PtdIns(4,5)P(2). Remarkably, NMR spectroscopy studies suggested differences in lipid-binding mechanisms. In addition, we experimentally demonstrated that sulfatide- and PtdIns(4,5)P(2)-binding sites overlap in Dab2 N-PTB and that both lipids stabilize the protein against temperature-induced unfolding. We found that whereas sulfatides induced conformational changes and facilitated Dab2 N-PTB penetration into micelles, Dab2 N-PTB bound to PtdIns(4,5)P(2) lacked these properties. These results further support our model that platelet membrane sulfatides, but not PtdIns(4,5)P(2), protect Dab2 N-PTB from thrombin cleavage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Disabled-2 (Dab2) inhibits platelet aggregation by competing with fibrinogen for binding to the α(IIb) β(3) integrin receptor, an interaction that is modulated by Dab2 binding to sulfatides at the outer leaflet of the platelet plasma membrane. The disaggregatory function of Dab2 has been mapped to its N-terminus phosphotyrosine-binding (N-PTB) domain. Our data show that the surface levels of P-selectin, a platelet transmembrane protein known to bind sulfatides and promote cell-cell interactions, are reduced by Dab2 N-PTB, an event that is reversed in the presence of a mutant form of the protein that is deficient in sulfatide but not in integrin binding. Importantly, Dab2 N-PTB, but not its sulfatide binding-deficient form, was able to prevent sulfatide-induced platelet aggregation when tested under haemodynamic conditions in microfluidic devices at flow rates with shear stress levels corresponding to those found in vein microcirculation. Moreover, the regulatory role of Dab2 N-PTB extends to platelet-leucocyte adhesion and aggregation events, suggesting a multi-target role for Dab2 in haemostasis., (© 2011 Blackwell Publishing Ltd.)

Background: Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for alphaIIbbeta3 integrin receptor binding by an unknown mechanism., Methodology/principal Findings: Using protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB), specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (K(d)) of 0.6 microM as estimated by surface plasmon resonance (SPR) analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process., Conclusions/significance: Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.