The structure and dynamics of the granulocyte macrophage colony-stimulating factor receptor defined by the ternary...

Myeloid cell lines and primary leukemic myeloblasts express two classes of granulocyte macrophage colony-stimulating factor (GM-CSF) binding sites of high (Kd 20-50 pM) and low affinity (Kd 5-10 nM). High affinity binding is caused by the association of two chains, p80 alpha and p130 beta, whereas p80 alpha alone confers low affinity binding only. Furthermore interleukin-3 (IL-3) competes for the binding of GM-CSF to its high affinity receptor (for review see Nicola, N. A., and Metcalf, D. (1991) Cell 67, 1-4). In the present study, we took advantage of the perturbation of GM-CSF binding equilibrium by IL-3 to take a quantitative approach to analysis of the structure and dynamics of the GM-CSF receptor complex. First, cross-linking studies were performed at two concentrations of radioligand. At 200 pM, a concentration sufficient for near saturation of the high affinity binding site R1, the association between p80 alpha and p130 beta is stoichiometric, and the addition of IL-3 prevents the binding to both chains. At 5 nM, a concentration sufficient for half-occupancy of the low affinity binding site R2, IL-3 prevents cross-linking to the beta chain only. Second, GM-CSF saturation curves were analyzed both at equilibrium and under conditions of perturbation of the equilibrium by IL-3. In the presence of IL-3, the interaction of GM-CSF with its receptor is converted from high to low affinity binding. Computer modeling of binding data with a ternary complex model involving GM-CSF, p80 alpha, and p130 beta indicates that the model fits the data with accuracy and suggests that ligand binding stabilizes the interaction between p80 alpha and p130 beta by 3 orders of magnitude. Third, membrane solubilization dissociates p80 alpha and p130 beta whereas on ligand-stabilized preformed complexes, solubilization did not dissociate the two chains. Finally, upon addition of GM-CSF, there is an increase with time in the proportion of ligand bound to the high affinity receptor, at the expense of that bound to low affinity receptor, suggesting that stabilization of the ternary complex is a time-dependent process.