title: Lipid-mediated membrane binding properties of Disabled-2.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Ruba
authors.initials: R
authors.lastname: Alajlouni
authors.forename: Karen E
authors.initials: KE
authors.lastname: Drahos
authors.forename: Carla V
authors.initials: CV
authors.lastname: Finkielstein
authors.forename: Daniel G S
authors.initials: DG
authors.lastname: Capelluto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D002942:Circular Dichroism; D008081:Liposomes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013433:Sulfoglycosphingolipids / Q000737:chemistry; D020349:Surface Plasmon Resonance; D025521:Tumor Suppressor Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C102885:DAB2 protein, human; D008081:Liposomes; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D013433:Sulfoglycosphingolipids; D025521:Tumor Suppressor Proteins