The cell surface protease receptor tissue factor (TF) supports monocyte migration, metastasis and angiogenesis by mechanisms attributed to signaling functions of its cytoplasmic domain. The presented preliminary data demonstrate that TF supports cell adhesion, migration and intracellular signaling through a specific molecular pathway mediated by recruitment of actin-binding protein 280 (ABP-280) to the TF cytoplasmic tail. This application proposes to analyze the details of TF signaling.
Aim 1 characterizes the interaction of the TF cytoplasmic domain with the carboxyl- terminus of ABP-280. Site-directed mutagenesis experiments define the molecular recognition motif in TF for ABP-280, surface plasmon resonance analyses with purified proteins address the importance of TF dimerization for ABP-280 binding, and transfection studies with ABP-280 deficient cell lines serve to demonstrate that the carboxyl-terminus of ABP-280 is required for TF interaction and signaling.
Aim 2 analyzes the proximal signaling events that follow TF ligation by characterizing phosphorylation of TF and ABP-280. ABP-280 is hypothesized to mediate recruitment of adaptor or signaling molecules to TF adhesion contacts. Yeast two hybrid screening and a knowledge guided search are proposed as strategies to identify these additional elements of the signaling pathway.
Aim 3 analyzes effector functions of small GTPases in TF-initiated signaling. By transfection experiments with dominant negative mutants, by the use of pathway blocking inhibitors, and by the analysis of rac activation, support is established for the hypothesis that the GTPases rac and downstream effectors are responsible for the ruffle-like actin organization induced by TF-ligation. The fundamental knowledge and the tools developed by these studies will aid the design of experimental strategies that define biological functions of TF in more complex scenarios in vivo. In the long term, elucidation of the molecular details of TF signaling will guide novel therapeutic approaches that intervene with functions of TF in metastasis and angiogenesis.
Showing the most recent 10 out of 40 publications
