Separate Functions for the Two Modules of the Membrane-proximal Cytokine Binding Domain of Glycoprotein 190, the Leukemia Inhibitory Factor Low Affinity Receptor, in Ligand Binding and Receptor Activation

Mathieu-Benoı̂T Voisin,Juliette Bitard,Sophie Daburon,Jean-François Moreau,Jean-Luc Taupin

doi:10.1074/jbc.m111624200

Abstract

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each. 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 immunoglobulin-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin-like repeats. A minimal D1IgD2 fragment is required for binding LIF. By using transmembrane forms of deletion mutants in gp190 ectodomain, we demonstrated that removal of D1 led to spontaneous activation of the receptor and that this property was devoted to a peptidic sequence localized within the last 42 amino acids of the carboxyl-terminal module of D2. By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2. Therefore, the two constitutive modules of the membrane-proximal CRH domain D2 of gp190 fulfill two distinct roles in gp190 function, i.e. in stabilizing the conformation of gp190 allowing LIF binding and in activating the receptor.

Highlights

The leukemia inhibitory factor (LIF)[1] low affinity receptor gp[190] belongs to the large family of the hematopoietic recep
The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp[190] and the high affinity converter gp[130]. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each
By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2

Summary

EXPERIMENTAL PROCEDURES

Monoclonal Antibodies and Cytokines—The anti-gp[190] mAbs specific for domains D1 (6G8) or D2 (12D3, 8C2) have already been described (9, 10). Cell Transfection Experiments—Parent Ba/F3 cells were maintained in RPMI supplemented with 8% fetal calf serum, 2 mM L-glutamine, and murine IL-3 at a final 1/200 dilution They were washed and electroporated (280 V, 900 microfarads, infinite resistance) using an Easyjectϩ electroporator (Eurogentec, Seraing, Belgium), with plasmids pRcglo or pRcglo-gp[130], which bore the neomycin resistance gene, in the presence or absence of the pEDr plasmid containing either the gp190/130 chimera or the chimeric transmembrane deletion mutants of the gp[190] ectodomain. All the gp[190] deletion mutants we used in this study were cloned in the pEDr plasmid, which contains a functional DHFR encoding sequence that confers to the transfected CHO-DUCKX cells, the ability to grow in the nucleoside-free selection medium. After neutralization of trypsin with 1:10 volume of serum, fMTX-DHFR intracellular complexes were analyzed by flow cytometry

RESULTS

Conditions for transfection and cytokine selectiona

Autonomous proliferation of

DISCUSSION