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Abstract
The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.
Highlights
The leukemia inhibitory factor (LIF)1 low affinity receptor gp190 belongs to the large family of the hematopoietin receptors, which are characterized by a consensus cytokine receptor homology (CRH) domain
Our alanine-scanning analysis of the carboxyl terminus of the Ig-like domain of hgp190 showed that mutation to alanine of 6 of 8 residues impaired human LIF (hLIF) binding in our ELISA assay, FIG. 9
Part of the hLIF/hgp190 complexes can dissociate during this 1.5 h time lapse and the loss in the ELISA signal as compared with equilibrium conditions will be exponentially related to the kinetic dissociation constant of these complexes
Summary
Cell Culture—All eukaryotic cell lines were grown in 5% CO2 at 37 °C in a water-saturated atmosphere either in Dulbecco’s modified Eagle’s medium (Invitrogen) for COS and CHO DUCKX cells or RPMI 1640 medium for Ba/F3 cells, in the presence of 8% fetal calf serum (Invitrogen) and 2 mM L-glutamine. To analyze the turnover of the transmembrane form of the LIF receptor or its mutants from the cell surface, the indicated Ba/F3 cell lines were incubated with hLIF at 100 ng/ml or without any added cytokine for the indicated times at 37 °C, before being stained with the anti-hgp190 8C2 mAb or an isotype-matched negative control mAb. ELISAs for the Quantitation of shgp190 and of the LIF/shgp190 Complexes—Secreted shgp190myc and its mutants were quantitated using a sandwich ELISA specific for hgp190, according to the previously reported procedure [12, 13]. CHO-derived hLIF at a final concentration of 10 ng/ml giving maximal STAT3 phosphorylation was incubated with or without the purified soluble receptors, or with normal mouse serum as a source of mLIFR, at twice the indicated concentrations at 37 °C for 20 min, before the cells (2 ϫ 105 per condition tested) were added in an identical volume.
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