Structural and functional studies of GM-CSF and IL-3 receptor systems
Creators: name: Mirza, Shamaruh
Publisher: name: The Australian National University
Year: 2010
Subjects: subject: QR185.8.C95 M57 2010
subject: Granulocyte-macrophage colony-stimulating factor
subject: Interleukin-3
subject: Cytokines Receptors

description: The cytokines interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony stimulating factor (GM-CSF) exhibit overlapping activities in the regulation of hematopoietic cells. This thesis explores IL-3 and GM-CSF receptor binding and activation mechanisms. GM-CSF is an important mediator of inducible hematopoiesis and inflammation with a critical role in the function of alveolar macrophages. GM-CSF signals via GM-CSFR{u03B1} and h{u03B2}c. This thesis investigated the role of the Ig-like domain of the GM-CSFR{u03B1} in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of h{u03B2}c. Val{u2075}{u00B9} and Cys{u2076}{u2070} were found having critical roles in binding to the {u03B1} receptor with Arg{u2075}{u2074} and Leu{u2075}{u2075} also important. High affinity binding in the presence of h{u03B2}c was strongly affected by mutation of Cys{u2076}{u2070} and also reduced by mutation of Val{u2075}{u00B9} , Arg{u2075}{u2074} and Leu{u2075}{u2075}. Growth signalling was most severely affected by mutation of Cys{u2076}{u2070}. The results indicated a previously unrecognized role for the Ig-like domain, and in particular Cys{u2076}{u2070}, of GM-CSFR{u03B1} in the binding of GM-CSF and subsequent activation of cellular signalling. IL-3 is also a critical regulator of inflammation and immune responses in mammals. It exerts its effects on target cells via receptors comprising IL-3R{u03B1}-subunit and h{u03B2}c or a {u03B2}-subunit that specifically binds IL-3 ({u03B2}IL-3; only in mice). Two splice variants of IL-3R{u03B1} have been recently identified which are relevant to haematopoietic progenitor cell differentiation or proliferation: the full length ("SPI" isoform) and a novel isoform lacking the N-terminal Ig-like domain (denoted "SP2"). This thesis demonstrated that each mIL-3R{u03B1} isoform can direct mIL-3 binding to two distinct sites on the {u03B2}IL-3 subunit. It also investigated which residues in mIL-3 itself are critical to the two modes of {u03B2}IL-3 recognition, and whether the human IL-3R{u03B1}SPI and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the h{u03B2}c subunit. It identified the residues clustering around the highly-conserved A-helix residue, Glu{u00B2}{u00B3} , in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the {u03B2}IL-3 and mIL-3R{u03B1}SP2 subunits, whilst an overlapping cluster was required for binding and activation of {u03B2}IL-3 in the presence of mIL-3R{u03B1}SP1. Subsequently, studies of human IL-3 indicated that two different modes of {u03B2}c binding are utilized in the presence of the hIL-3R{u03B1}SP1 or SP2 isoforms, suggesting a conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signalling outcomes. A widely accepted hypothesis is that h{u03B2}c receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in h{u03B2}c and unidentified cysteine residues in the {u03B1} receptors. This thesis also investigated whether the distortion of the crucial domain l-domain 4 ligand-binding epitope in h{u03B2}c and the related mouse receptor, {u03B2}IL-3, could account for the defects in receptor activation caused by disruption of the domain 1 D-E loop disulfide bond. Indeed, mutation of the D-E loop disulfide in h{u03B2}c led to a loss of high-affinity hIL-3 binding with an IL-3R{u03B1}SP2 and a consequent loss of downstream signalling. Mutation of the orthologous residues in {u03B2}IL-3, precluded direct binding of mIL-3 and also resulted in complete loss of high-affinity binding and downstream signal transduction when coexpressed with a mIL-3R{u03B1}SP2 isoform. These data were most consistent with a role for the h{u03B2}c domain 1 D-E loop disulfide, and the orthologous residues in the {u03B2}IL-3 receptor, in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signalling.