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 t o the superfamily of cytokine receptors characterized by a common stru ctural feature, i.e., the presence of at least two fibronectin-like fo lds in the extracellular domain, which was first identified in the gro wth hormone receptor. The GM-CSF receptor (GMR)-alpha chain confers lo w affinity binding only (5-10 nM), whereas the other chain, beta c, do es not bind GM-CSF by itself but confers high affinity binding when as sociated 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 elect rostatic interact-ion between these two residues. Through site directe d mutagenesis, we provide several Lines of evidence indicating the imp ortance of electrostatic interaction in ligand-receptor recognition. F irst, 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 hi gh (26 pM) toward low affinity (from 2 to 13 nM). The rank order of th e dissociation constant for the different GMR-alpha R280 mutations whe re Lys > Gin > Met > Asp, suggesting the importance of the charge at t his position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in recept or 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 ra te constant and only a 2-fold change in the dissociation rate constant , suggesting that R280 is implicated in ligand recognition, possibly t hrough interaction with Asp112 of GM-CSF. For all R280 mutants, the ha lf-efficient concentrations of GM-CSF required for membrane (receptor binding) to nuclear events (c-fos promoter activation) and cell prolif eration (thymidine incorporation) were in the same range, indicating t hat the threshold for biologic activity is governed mainly by the affi nity of ligand-receptor interaction. Furthermore, mutation of other re sidues in the immediate vicinity of R280 was less drastic. Sequence al ignment and modeling of interleukin (IL)-3R and IL-5R identified an ar ginine 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 s imilar to the one described herein for GMR.