Stimulation of platelets by a wide variety of agonists transforms alpha-IIb-beta3 from its resting state to an active conformation, in which it is capable of serving as a receptor for fibrinogen or von Willebrand Factor in a divalent ion dependent interaction. Engagement of these ligands supports platelet aggregation, a critical event for hemostasis and also the underlying event for the thrombotic diseases. At a molecular level, the transformation of alpha-IIb-beta3 from a resting to an active state requires the generation of an inside-out signal, which is initiated at its intracellular face, composed of the cytoplasmic tails of the alpha-IIb and beta3 subunits, and transforms the extracellular face of the receptor to render it competent to bind ligand. The overall goal of this project is to understand the molecular basis for the generation of this inside-out signal at the cytoplasmic face of the receptor and for the regulatory role of divalent cations in ligand binding to the extracellular domain of alpha-IIb-beta3. Preliminary data, including a NMR structure, support the hypothesis that the inside-out signal is initiated by dissociation of the complex between the alpha-IIb and beta3 cytoplasmic tails, which permits insertion of these free tails into the membrane.
In Specific Aim I, this hypothesis and the role of regulatory residues within each subunit will be tested utilizing synthetic peptides, mutational, biophysical, structural, and functional analyses.
In Specific Aim II, the influence of the equilibrium between the associated and dissociated cytoplasmic tail complex in regulating ligand binding, the capacity of intracellular molecules to induce activation and the applicability of these models to other integrin will be investigated using structural and functional assays.
In Specific Aim III, we will characterize the cation binding properties of alpha-v-beta3 and alpha-IIb-beta3 by equilibrium dialysis; locate the two functional cation binding sites that regulate ligand binding by mutagenesis and establish their relationship to the cation binding sites observed in the crystal structure of alpha-v-beta3; and then define differences in the spatial relationships of the cation and ligand binding sites of the two beta3 integrins that contribute to the differential influence of cations on ligand binding to alpha-v-beta3 and alpha-IIb-beta3. Taken together, these studies will provide key insights into the way alpha-IIb-beta3 initiates and mediates the key functional response of platelet aggregation, provide information on the activation and ligand binding functions of all integrins, and establish more effective ways to design antithrombotics and other integrin targeted drugs.
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