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 factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity 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 reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.
Hood
W F
WF
Searle Research and Development, Pharmacia Corporation, 700 Chesterfield Parkway North, St. Louis, Missouri 63198, USA. william.f.hood@pharmacia.com
Feng
Y G
YG
Schilling
R J
RJ
Gokarn
Y
Y
Jarvis
C
C
Remsen
E E
EE
Shieh
J J
JJ
Joy
W
W
Klein
B K
BK
Polazzi
J O
JO
Welply
J K
JK
McKearn
J P
JP
Monahan
J B
JB
D001667:Binding, Competitive; D002850:Chromatography, Gel; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3; D007700:Kinetics; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000819:agonists* / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C404145:myelopoietin; D016179:Granulocyte Colony-Stimulating Factor