Inter-species chimeras of leukaemia 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 high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered 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 interaction 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 between hLIF and mLIF that are responsible for the different properties of these proteins.
Owczarek
C M
CM
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Layton
M J
MJ
Metcalf
D
D
Lock
P
P
Willson
T A
TA
Gough
N M
NM
Nicola
N A
NA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6; D007700:Kinetics; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D018121:Receptors, Cytokine / Q000378:metabolism*; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection
D018076:DNA, Complementary; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; C507838:Lif protein, mouse; C507841:Lifr protein, mouse; D008222:Lymphokines; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins