CONSTRUCTION AND BINDING-KINETICS OF A SOLUBLE GRANULOCYTE-MACROPHAGECOLONY-STIMULATING FACTOR-RECEPTOR ALPHA-CHAIN-FC FUSION PROTEIN

Citation
C. Monfardini et al., CONSTRUCTION AND BINDING-KINETICS OF A SOLUBLE GRANULOCYTE-MACROPHAGECOLONY-STIMULATING FACTOR-RECEPTOR ALPHA-CHAIN-FC FUSION PROTEIN, The Journal of biological chemistry, 273(13), 1998, pp. 7657-7667
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
273
Issue
13
Year of publication
1998
Pages
7657 - 7667
Database
ISI
SICI code
0021-9258(1998)273:13<7657:CABOAS>2.0.ZU;2-X
Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alph a-chain (GM-CSFR alpha), which specifically binds GM-CSF, and a beta-c hain (beta(c)), shared with the interleukin-3 and interleukin-5 recept ors, GM-CSFR alpha exists in both a transmembrane (tmGM-CSFR alpha) an d a soluble form (sGM-CSFR alpha). We designed an sGM-CSFR alpha-Fc fu sion protein to study GM-CSF interactions with the GM-CSFR alpha. The construct was pre pared by fusing the coding region of the sGM-CSFR al pha with the CH2-CH3 regions of murine IgG2a, Purified sGM-CSFR alpha- Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel elect rophoresis but formed a trimer of 160-200 kDa under nonreducing condit ions. The sGM-CSFR alpha-Fc bound specifically to GM-CSF as demonstrat ed by standard and competitive immunoassays, as well as by radioligand assay with I-125-GM-CSF, The sGM-CSFR alpha-Fc also inhibited GM-CSF- dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFR alpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems, In t he first, the sGM-CSFR alpha-Fc was bound to immobilized staphylococca l protein A on the biosensor surface, and binding kinetics of GM-CSF i n solution were determined, This revealed a rapid K-off of 2.43 x 10(- 2)/s, A second set of experiments was performed with GM-CSF immobilize d to the sensor surface and the sGM-CSFR alpha-Fc in solution, The dis sociation rate constant (k(off)) for the sGM-CSFR alpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of bin ding in this assay, These data indicate rapid dissociation of GM-CSF f rom the sGM-CSFR alpha-Fc and suggest that in vivo, sGM-CSFR alpha may need to be present in the local environment of a responsive cell to e xert its antagonist activity.