Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an ␣-chain (GM-CSFR␣), which specifically binds GM-CSF, and a ␤-chain (␤ c ), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFR␣ exists in both a transmembrane (tmGM-CSFR␣) and a soluble form (sGM-CSFR␣). We designed an sGM-CSFR␣-Fc fusion protein to study GM-CSF interactions with the GM-CSFR␣. The construct was prepared by fusing the coding

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... of the sGM-CSFR␣ with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFR␣-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-CSFR␣-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125 I-GM-CSF. The sGM-CSFR␣-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFR␣-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFR␣-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 k off of 2.43 ؋ 10 ؊2 /s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFR␣-Fc in solution. The dissociation rate constant (k off ) for the sGM-CSFR␣-Fc trimer from GM-CSF was 1.57 ؋ 10 ؊3 /s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFR␣-Fc and suggest that in vivo, sGM-CSFR␣ may need to be present in the local environment of a responsive cell to exert its antagonist activity.