Structural Insights into the Extracellular Assembly of the Hematopoietic FLT3 Ligand-Receptor Complex

Samenvatting

Fms-like tyrosine kinase 3 (Flt3) is a type III receptor tyrosine kinase (RTKIII) expressed at the cell surface of hematopoietic stem cells and early hematopoietic progenitors and initiates signaling pathways crucial for the development of the pluripotent cell population that gives rise to the cellular repertoire of the human blood and immune systems. The Flt3 receptor is activated by the four helical bundle cytokine termed Flt3 Ligand (FL). The aim of the research described in this work was to obtain molecular insights into the first key step of this activation process, namely the binding of the ligand to the extracellular region of its receptor, by methods as X-ray crystallography, small-angle X-ray scattering, negative-stain electron microscopy and isothermal titration calorimetry.
In the first part of this thesis, Chapter 1, the existing scientific knowledge about RTKIII activation is reviewed. The chapter starts with an introductory section on the hematopoietic system and the diverse types of tyrosine kinase receptors. Next, the molecular properties of the Flt3 receptor and its ligand are discussed and the homology with the other ligands and receptors in the RTKIII and RTKV family is highlighted. Then, following a short section about the important function of the Flt3 system in hematopoiesis, a detailed overview of the landmark experiments that lead to the current understanding of ligand-induced receptor activation in the RTKIII and closely related RTKV family is presented. The chapter ends with a discussion on the activation mechanism of the intracellular tyrosine kinase domains for both wild-type and oncogenic Flt3.
Chapters 2, 3 and 4 present the results obtained during this doctoral research. In the first stage of the project, protocols for the efficient recombinant production of FL and Flt3 ectodomains were established. As clarified in Chapter 2, a bioactive recombinant form of soluble human FL could be obtained by in vitro refolding inclusion bodies that were produced in E.coli. Chapter 3 describes the production of Flt3 receptor ectodomain variants as secreted proteins in inducible mammalian cell lines, and the crystallization of Flt3 ligand-receptor complexes for structural studies by X-ray crystallography.
In Chapter 4, the acquired structural and thermodynamic insights into the molecular mechanism that underlies the human Flt3 ligand-receptor interaction are presented, with a focus on the determined X-ray structure of a ternary Flt3 ligand-receptor complex. Finally, in Chapter 5, the obtained results are discussed and suggestions for future research are present.

Datum prijs	11 mrt. 2011
Originele taal	English
Begeleider	Remy Loris (Jury) & Savvas Savvides (Promotor)