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-G Loop of the
Human Granulocyte/Macrophage Colony-stimulating Factor
Receptor 
Chain for Ligand Recognition
By
§
¶
From the * Clinical Research Institute of Montréal, Laboratory of Hemopoiesis and Leukemia, and The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed
of two chains, Laboratory of Biological Chemistry, Montréal, Quebec H2W 1R7, Canada; § Department of
Pharmacology, Department of Biochemistry, and ¶ Department of Molecular Biology, University of
Montréal, Montréal, Quebec H3C 3J7, Canada; ** The Genetics Institute, Cambridge,
Massachusetts 02140; and Hanson Cancer Center, Adelaide 5000, Australia
and 
c. Both chains belong to the superfamily of cytokine receptors characterized by a common structural feature, i.e., the presence of at least two fibronectin-like folds
in the extracellular domain, which was first identified in the growth hormone receptor. The
GM-CSF receptor (GMR)- chain confers low affinity binding only (5-10 nM), whereas the
other chain, c, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR- (25-100 pM). The present study was designed to define the assembly of the GMR complex at the molecular level through site-directed mutagenesis guided by homology modeling with the growth hormone receptor complex. In our three-dimensional model,
R280 of GMR-, located in the F
-G loop and close to the WSSWS motif, is in the vicinity
of the ligand Asp112, suggesting the possibility of electrostatic interaction between these two
residues. Through site directed mutagenesis, we provide several lines of evidence indicating the importance of electrostatic interaction in ligand-receptor recognition. First, mutagenesis of
GMR-R280 strikingly ablated ligand binding in the absence of common (c); ligand binding was restored in the presence of c with, nonetheless, a significant shift from high (26 pM)
toward low affinity (from 2 to 13 nM). The rank order of the dissociation constant for the different GMR-R280 mutations where Lys > Gln > Met > Asp, suggesting the importance of
the charge at this position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold). Third, removal of the
charge at position R280 of GMR- introduced a 10-fold decrease in the association rate constant and only a 2-fold change in the dissociation rate constant, suggesting that R280 is implicated in ligand recognition, possibly through interaction with Asp112 of GM-CSF. For all
R280 mutants, the half-efficient concentrations of GM-CSF required for membrane (receptor
binding) to nuclear events (c-fos promoter activation) and cell proliferation (thymidine incorporation) were in the same range, indicating that the threshold for biologic activity is governed
mainly by the affinity of ligand-receptor interaction. Furthermore, mutation of other residues
in the immediate vicinity of R280 was less drastic. Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a turn in a highly divergent context at the F-G loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein
for GMR.
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