Leukemia inhibitory factor (LIF), cardiotrophin-1, and oncostatin M share structural binding determinants in the immunoglobulin-like domain of LIF receptor.
Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.
Plun-Favreau
Hélène
H
INSERM U564, Centre Hospitalier Universitaire d'Angers, 4 rue Larrey, 49033 Angers, France.
Perret
David
D
Diveu
Caroline
C
Froger
Josy
J
Chevalier
Sylvie
S
Lelièvre
Eric
E
Gascan
Hugues
H
Chabbert
Marie
M
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D016207:Cytokines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006131:Growth Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6; D007700:Kinetics; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053683:Oncostatin M; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053606:Receptors, OSM-LIF; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014162:Transfection
D016207:Cytokines; D006131:Growth Inhibitors; D007136:Immunoglobulins; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D046911:Macromolecular Substances; C508351:OSM protein, human; D010455:Peptides; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011994:Recombinant Proteins; D053683:Oncostatin M; C092073:cardiotrophin 1