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Abstract
Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg288 in the F'-G' loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor.
Highlights
The granulocyte colony-stimulating factor receptor (G-CSFR)1 is a member of the class 1 cytokine receptor family which is defined by the presence of a cytokine receptor homology (CRH) domain that is characterized by four conserved Cys residues and a conserved WSXWS (Trp-Ser-X-Trp-Ser, where X is any amino acid) sequence [1]
Between the sequences of the CRH domains of Granulocyte colony-stimulating factor (G-CSF) receptor and growth hormone receptor we found a 34% sequence similarity and 13% sequence identity, which is in a range where homology modeling is difficult, but still feasible
Site-directed mutagenesis of cytokine receptors, in which selected residues are replaced to evaluate the contribution of the amino acid side chains to ligand binding, has provided information about the binding sites of many receptors in this family (39 – 45)
Summary
G-CSF-R, granulocyte colony-stimulating factor receptor; BC, C-terminal ligand binding; BN, N-terminal ligand binding; CRH, cytokine receptor homologous; EPO-R, erythropoietin receptor; FnIII, fibronectin type III; GH-R, growth hormone receptor; mAb, monoclonal antibody; IL, interleukin. Bazan [1] proposed that the conserved CRH domain is comprised of two modules of fibronectin type III (FnIII)-like structure, each containing seven -strands which form two -sheets This model has been confirmed by determination of the crystal structures of several receptors including the growth hormone receptor (GH-R) [9], prolactin receptor [10], erythropoietin receptor (EPO-R) [11], and related class 2 cytokine receptors: tissue factor [12] and interferon-␥ receptor ␣ [13]. We have modeled the structure of the G-CSF-R CRH domain on the GH-R structure [9] and predicted regions likely to be involved in ligand binding These regions have been substituted with alanine residues to determine their contribution to ligand binding and receptor function.
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