Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist

Myelopoietins (MPOS) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating fact or (G-CSF) receptor agonists. In this study, MPO molecules containing one o f three different IL-3 receptor agonists linked with a common G-CSF recepto r agonist have been examined for their IL-3 receptor binding characteristic s. Binding to the alpha -subunit of the IL-3 receptor revealed that the aff inity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was refle cted in the MPO chimeras having approximately 2-fold slower, dissociation r ates and 2.7-5.5-fold slower association rates than the corresponding speci fic IL-3 receptor agonists alone. The affinity of binding of the MPO molecu les to the heteromultimeric alpha beta IL-3 receptor expressed on TF-1 cell s was either 3-, 10-, or 42-fold less potent than that of the individual co gnate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonan ce, near-UV circular dichroism, dynamic light scattering, analytical ultrac entrifugation, and size exclusion chromatography experiments determined tha t there were significant tertiary structural differences between the MPO mo lecules. These structural differences suggested that the IL-3 and G-CSF rec eptor agonist domains within the MPO chimera may perturb one another to var ying degrees. Thus, the differential modulation of affinity observed in IL- 3 receptor binding may be a direct result of the magnitude of these interdo main interactions.