Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

William F. DeGrado

Abstract

On circulating platelets, the integrin fibrinogen receptor &alphaIIb&beta3 favors inactive conformations. Platelets rapidly activate &alphaIIb&beta3 to bind fibrinogen, mediating a platelet clot. Resting &alphaIIb&beta3 is stabilized by interactions between the &alphaIIb and &beta3 transmembrane domains. Binding of talin-1 and kindlin-3 to the integrin cytoplasmic domain stabilizes separation of the TMs and receptor activation. Src family kinases are needed for transmission of extracellular signals into the cell. We have sought to better understand how signals are transmitted across the &alphaIIb&beta3 TM domain. First we characterized the structure and dynamics of the active and inactive integrin cytoplasmic domain to determine how motifs that bind talin-1 and kindlin-3 are affected by the integrin activation state and the membrane environment. The &alphaIIb&beta3 cytoplasmic domain is disorded, while the &beta3 subunit contains two &alpha-helices, which interact with the phospholipid bilayer. The close proximity of &alphaIIb to &beta3 in the inactive state induces a kink that projects the &beta-chain parallel to the membrane surface. This kink is likely to stabilize interactions between helices in &beta3 with the membrane and possibly compete with binding to talin-1 and kindlin-3. The &beta3 cytoplasmic domain becomes increasingly dynamic further from the membrane, suggesting that distal kindlin-3 binding residues should be more accessible to interact with their binding partners. We also studied the interaction between talin-1 and the &beta3 cytoplasmic domain to determine how the membrane environment affects formation of the talin-1/&beta3 complex. The membrane significantly increases the affinity of talin-1 for &beta3, but that the ternary integrin/talin-1/membrane complex is not significantly more stable than the talin-1/membrane complex alone. We also performed preliminary experiments characterizing the interaction between &alphaIIb&beta3 and both kindlin-3 and c-Src. Unlike talin-1, kindlin-3 does not require the membrane to bind the integrin cytoplasmic domain with high affinity. The interaction between c-Src and the integrin was extremely weak suggesting that it might require colocalization through other interactions. Finally, we developed a simplified model showing how thermodynamic coupling between integrin subunits might allow &alphaIIb&beta3 to adopt multiple activation states.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS