The objective of this project is to correlate the structure and function of the platelet membrane integrin allb|33. allb|33 is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and von Willebrand factor is exposed by platelet stimulation. Ligand binding to cdlb|33 is responsible for platelet aggregation and is a critical step in the formation of hemostatic platelet plugs and pathologic arterial thrombi. Integrins like allb|33 reside on cell surfaces in an equilibrium between low affinity (inactive) and high affinity (active) conformations. We have reported that integrin transmembrane domains engage in both specific heteromeric and homomeric interactions that define their inactive and active states, respectively and have proposed a """"""""push-pull"""""""" hypothesis to explain how integrin activity is regulated. Thus, processes that stabilize the active conformation of allb|33 would push it toward to its activated state, whereas processes that are more favorable when the transmembrane domains separate would pull the equilibrium in the same direction.
The Aims of the project will further characterize the """"""""push-pull"""""""" hypothesis.
In Aim 1, we will identify and characterize the interface that mediates the homomeric and heteromeric association of the (33 transmembrane helix, examine the structural basis for the specificity of integrin transmembrane domain interactions, and determine how changes in the relative positions of the allb and (33 transmembrane domains alter the allb(33 activation state.
Aim 2 will examine the contribution of transmembrane domain separation and oligomerization to the interaction of allb|33 with cytoplasmic proteins, focusing on the interaction of the (33 cytoplasmic domain with the cytoskeletal protein talin. We will use a recently developed tethered lipid membrane surface plasmon resonance system to study the interactions of the (33 cytoplasmic domain, talin, and phospholipids in a native membranelike environment. NMR structures for heteromeric and homomeric complexes of the allb and (33 transmembrane and cytoplasmic domains will be obtained as well.
In Aim 3, we will use our recently modified laser tweezers system to measure the lifetime of cdlb|33-ligand bonds, enabling us to derive quantitative thermodynamic and kinetic information about the nature of this interaction.
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