The Structural Basis for Interaction Between Colony-Stimulating Factor-1 and Its Receptor, CSF-1R

Hamwood, Tamarind (2007). The Structural Basis for Interaction Between Colony-Stimulating Factor-1 and Its Receptor, CSF-1R PhD Thesis, Institute for Molecular Bioscience, University of Queensland.

       
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Author Hamwood, Tamarind
Thesis Title The Structural Basis for Interaction Between Colony-Stimulating Factor-1 and Its Receptor, CSF-1R
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Professor David Hume
Abstract/Summary Macrophages are white blood cells involved in many aspects of host defence, immunity, pathology and homeostasis. Colony-stimulating factor-1 (CSF-1) is required for normal proliferation, differentiation and maturing of macrophage lineage cells. CSF-1 binds to its proto-oncogene receptor, CSF-1R (also known as c-fms) to induce phosphorylation of cytoplasmic tyrosine residues on the receptor and activation, which initiates molecular signalling cascades leading to specific gene regulation. Given the central role of macrophages in immunity and pathology, CSF-1 and its receptor are obvious drug targets. This study involves the structural characterization of the interaction between CSF-1 and its receptor with the eventual aim of designing novel therapeutics to act antagonistically on CSF-1R. Three approaches were taken towards this aim. In Chapter 3, molecular modelling and inter-species cross-reactivity of CSF-1 was used to identify residues likely to be involved in the interaction between CSF-1 and its receptor. The crystal structure of human CSF-1 (PDB code 1hmc), a four-helix bundle cytokine, was used to create a model of murine CSF-1. The CSF-1R is a type III receptor tyrosine kinase (RTK), with five extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane region and a cytoplasmic kinase domain. The ligand-binding regions, located within the three N-terminal Ig-like domains of the receptor, were modelled on similar cell-surface receptors. An investigative comparison of mouse and human structural models identified seven residues on mouse CSF-1R as candidates for interaction with CSF- 1 based on structural location, solvent exposure, electrostatic complementarity, evolutionary conservation and prevalence in protein-protein interaction sites. To further explore these residues, mutated CSF-1R constructs were assayed in two independent systems. The first assay utilized endogenous signalling by CSF-1R that leads to transcriptional activation of urokinase plasminogen-activator (uPA). Mutant or wild type CSF-1R constructs were co-transfected with a luciferase reporter construct under regulation of the uPA promoter. Luciferase activity measured CSF-1-dependent CSF-1R signalling, to identify mutant receptors not capable of responding to CSF-1. Mutant receptors were also assayed for the ability to promote CSF-1-induced growth in the Ba/F3 cell line. Evidence from both assays confirmed that R127, Y238 and W140 are essential for CSF-1-induced activation of the receptor. Due to the complex disulfide-linkages and glycosylation patterns of CSF-1 and CSF-1R, obtaining crystallization amounts of pure protein required empirical study of different expression systems. As the interaction does not require the transmembrane domain, only the ligand-binding regions of murine CSF-1R were included in the his-tagged constructs. The first contained the entire extracellular region (F15), while shorter truncations contained either the N-terminal three domains (F13) or the second and third Ig-like domains (F23). In Chapter 4, these constructs and CSF-1 were expressed in bacteria as inclusion bodies, purified to homogeneity by metal-affinity chromatography and refolded in the presence of disulfide-exchange factors. While CSF-1 refolded with little aggregation, receptor proteins showed significant aggregation upon removal of denaturants. To overcome aggregation during refolding, receptor constructs were cloned into a baculovirus vector for expression as a secreted protein in a eukaryotic insect cell system. In Chapter 5, insect cell production of CSF-1R proteins for large-scale production was optimised. Chapter 6 describes various assays for protein integrity and activity that were undertaken on the proteins produced in Chapters 4 and 5. In particular, the native activity of CSF-1 to promote bone marrow macrophage growth was examined, as was the ability of soluble receptor and neutralizing antibody to prevent this activity. Additionally, an assay amenable to high-throughput screening for agents that interact specifically with the extracellular domains of CSF-1R was developed in the Ba/F3 cell line. This assay shows excellent dosedependent response to CSF-1, with maximal activity double the baseline activity, and may be used to identify both agonists and antagonists of the interaction.

 
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