Crucial role of the residue R280 at the F'-G' loop of the human granulocyte/macrophage colony-stimulating factor receptor alpha chain for ligand recognition

The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed of two chains, alpha and beta 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)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, beta c, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (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-alpha, 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 interact-ion 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-alpha R280 strikingly ablated ligand binding in the absence of beta common (beta c); ligand binding was restored in the presence of beta 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-alpha R280 mutations where Lys > Gin > 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-alpha 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 beta 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.