Construction and binding kinetics of a soluble granulocyte-macrophage colony-stimulating factor receptor alpha-chain-Fc fusion protein.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha). We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha. The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 x 10(-2)/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.
Monfardini
C
C
Department of Medicine, Rheumatology Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Ramamoorthy
M
M
Rosenbaum
H
H
Fang
Q
Q
Godillot
P A
PA
Canziani
G
G
Chaiken
I M
IM
Williams
W V
WV
D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D003001:Cloning, Molecular; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D007141:Immunoglobulin Fc Fragments; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor