Binding of leukemia inhibitory factor (LIF) to mutants of its low affinityreceptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains

The gp190 transmembrane protein,the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors c haracterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proxi mal CBD (herein called D2) is separated from the membrane-distal one (calle d D1) by an immunoglobulin-like (Ig) domain and is followed by three fibron ectin type III repeats. We used truncated gp190 mutants and a blocking anti -gp190 monoclonal antibody to study the role of these repeats in low affini ty receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D 1, suggesting functionally important interactions between the two CBDs in t he wild-type protein. In addition, point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest t hat at least two distinct sites, both located within the D1Ig region, are i nvolved in LIF binding to gp190, and more generally, that Ligand binding si tes on these receptors may well be located outside the canonical CBDs.