The objective of this project is to correlate the structure and function of the platelet membraneintegrin allb|33. allb|33 is a calcium-dependent heterodimer whose binding site for ligands such asfibrinogen and von Willebrand factor is exposed by platelet stimulation. Ligand binding to cdlb|33 isresponsible for platelet aggregation and is a critical step in the formation of hemostatic plateletplugs and pathologic arterial thrombi. Integrins like allb|33 reside on cell surfaces in an equilibriumbetween low affinity (inactive) and high affinity (active) conformations. We have reported thatintegrin transmembrane domains engage in both specific heteromeric and homomeric interactionsthat define their inactive and active states, respectively and have proposed a 'push-pull' hypothesisto explain how integrin activity is regulated. Thus, processes that stabilize the active conformationof allb|33 would push it toward to its activated state, whereas processes that are more favorablewhen 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 andcharacterize the interface that mediates the homomeric and heteromeric association of the (33transmembrane helix, examine the structural basis for the specificity of integrin transmembranedomain interactions, and determine how changes in the relative positions of the allb and (33transmembrane domains alter the allb(33 activation state.
Aim 2 will examine the contribution oftransmembrane domain separation and oligomerization to the interaction of allb|33 with cytoplasmicproteins, 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 tostudy the interactions of the (33 cytoplasmic domain, talin, and phospholipids in a native membranelikeenvironment. NMR structures for heteromeric and homomeric complexes of the allb and (33transmembrane and cytoplasmic domains will be obtained as well.
In Aim 3, we will use our recentlymodified laser tweezers system to measure the lifetime of cdlb|33-ligand bonds, enabling us toderive quantitative thermodynamic and kinetic information about the nature of this interaction.
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