INTERSPECIES CHIMERAS OF LEUKEMIA INHIBITORY FACTOR DEFINE A MAJOR HUMAN RECEPTOR-BINDING DETERMINANT

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF -R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual h igh-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hyb rids has been generated. Perhaps surprisingly, both of these propertie s mapped to the same region of the hLIF molecule. The predominant cont ribution was from residues in the loop linking the third and fourth he lices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted thre e-dimensional structure. Since all chimeras retained full biological a ctivity and receptor-binding activity on mouse cells, and there was li ttle variation in the specific biologic-al activity of the purified pr oteins, it can be concluded that the overall secondary and tertiary st ructures of each chimera were intact. This observation also implied th at the primary binding sites on mLIF and hLIF for the mLIF-R were unal tered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional i nteraction site to that used by both mLIF and hLIF to bind to the mLIF -R. These studies define a maximum of 15 amino acid differences betwee n hLIF and mLIF that are responsible for the different properties of t hese proteins.