Identification of Agonistic and Antagonistic Antibodies against gp190, the Leukemia Inhibitory Factor Receptor, Reveals Distinct Roles for Its Two Cytokine-binding Domains

Jean-Luc Taupin,Patrick Legembre,Juliette Bitard,Sophie Daburon,Vincent Pitard,Frédéric Blanchard,Laurence Duplomb,Anne Godard,Yannick Jacques,Jean-François Moreau

doi:10.1074/jbc.m105476200

Abstract

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130, both of which are members of the family of hematopoietic receptors characterized by the cytokine receptor homology (CRH) domain. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an Ig-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin repeats. We raised a series of monoclonal antibodies specific for the human gp190. Among them was the blocking antibody 1C7, which was directed against the D1Ig region and which impaired the binding of LIF to gp190. Another blocking antibody, called 12D3, was directed against domain D2 and interfered with the reconstitution of the high affinity receptor complex, independently of the interaction between LIF and gp190. The blocking effect of these two antibodies concerned four cytokines known to use gp190, i.e. LIF, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. Among 23 antibodies tested alone or in combination (two anti-D2 and 21 anti-D1Ig), only the mixture of the two anti-D2 antibodies displayed agonistic activity in the absence of the cytokine. Taken together, these results demonstrate that the two CRH domains of gp190 play different functions in ligand binding and receptor activation.

Highlights

The leukemia inhibitory factor (LIF)1 low affinity receptor gp190 belongs to the large family of the hematopoietic receptors that are characterized by a consensus cytokine receptor homology (CRH) domain, the so-called cytokine-binding do
D1 and D2 are separated by an Ig-like module of around 100 amino acids, and D2 is followed by three 100-amino acid-long type III fibronectin modules, the FN region [1]. gp190 participates in the high affinity receptor complex for five human cytokines, namely LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and the very recently discovered neurotrophin-1/B cell-stimulating factor 3 [3]
Anti-human gp190 mAb 1C7 and 12D3 Abrogate the Proliferation Induced by LIF, OSM, CT-1, and CNTF—We have generated 35 monoclonal antibodies specific for the human gp190 cytokine receptor, some of them having already been described [10]

Summary

EXPERIMENTAL PROCEDURES

Monoclonal Antibodies—The anti-gp190 mAb 12D3 was produced in the laboratory according to the protocol described in Ref. 10. Obtaining the chimeric transmembrane gp190/130 consisting of the extracellular region of gp190 fused to the transmembrane and intracellular domains of gp130, the generation of LIF-dependent Ba/F3 cell lines expressing wild-type gp130 and gp190 (Ba/F3 gp190 ϩ gp130), or gp130 and chimeric gp190/130 receptors (Ba/F3 gp190/130 ϩ gp130) has been described previously [7] These cell lines were maintained in RPMI supplemented with 8% fetal calf serum, 2 mM L-glutamine, and 50 ng/ml CHO-derived recombinant human LIF. The plastic-adherent human choriocarcinoma JAR cell line was cultured in Dulbecco’s modified Eagle’s medium supplemented with 8% fetal calf serum, harvested by treatment with trypsin, washed three times, and resuspended in PBS containing 0.5% bovine serum albumin. The mAbs were labeled with iodine, and the experiments were conducted essentially as previously described [13]

RESULTS

95 Ϯ 9 93 Ϯ 40

DISCUSSION